MÉTODO DE OTIMIZAÇÃO DE CÓDON DE UMA SEQUÊNCIA POLINUCLEOTÍDICA, POLINUCLEOTÍDEO, MÉTODOS PARA DISTRIBUIR UMA SEQUÊNCIA POLINUCLEOTÍDICA, PARA EXPRESSAR UM TRANSCRITO DE MRNA, PARA EXPRESSAR UMA PROTEÍNA E PARA TRATAR UM SUJEITO QUE TEM OU ESTÁ EM RISCO DE DESENVOLVER UMA DOENÇA E KITS

Abstract

Featured are methods and applications of codon optimization of a gene product. In particular, the disclosure features methods of codon optimization by reducing the frequency of m6A modifications so as to promote increased mRNA half-life and stability for the purpose of enhancing protein production. Additional methods of delivering codon optimized gene products are disclosed. The methods of the disclosure are clinically relevant for gene and cellular therapies and vaccine development.

Description

1 / 121 “CODON OPTIMIZATION METHOD FOR A POLYNUCLEOTIDE SEQUENCE, POLYNUCLEOTIDE, METHODS FOR DISTRIBUTING A polynucleotide sequence, to express an mRNA transcript, to express a protein, and to treat a subject who has or is at risk of developing "A DISEASE AND KITS"

[0001] This application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on March 1, 2024, is named “51772-004WO4_Sequence_Listing_3_1_24” and is 18,345 bytes in size. FIELD OF THE INVENTION

[0002] This disclosure relates to the field of nucleic acid engineering. Specifically, this disclosure provides compositions and methods for modifying the codon sequence of a nucleic acid encoding a polypeptide in order to enhance the expression of the desired polypeptide in an organism of interest, such as a human. FUNDAMENTALS

[0003] N(6)-methyladenosine (m6A), the most prevalent mRNA modification in mammals, influences broad aspects of gene expression in diverse physiological and pathophysiological processes. The METTL3-METTL14 methyltransferase complex installs m6A methylation in mRNA at a common DRACH sequence motif (D = A, G, or U; R = A or G; H = A, C, or U), but only a fraction of DRACH sequences (~5%) in a subset of cellular transcripts are selected for methylation. Furthermore, m6A exhibits a marked regional bias in its transcriptomic distribution, being strongly enriched in unusually long internal exons and those close to stop codons. Despite the central importance of Petition 870250096842, dated 10 / 23 / 2025, page 9 / 180 2 / 121 specific m6A deposition in m6A-mediated gene regulation, the mechanistic basis for m6A specificity remains poorly understood. There is still a need for compositions and methods that take advantage of this mechanistic basis to improve transgene expression in an organism of interest. SUMMARY OF THE INVENTION

[0004] In one aspect, the disclosure presents a method for optimizing codons in a polynucleotide sequence that encodes a polypeptide of interest. The method involves replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence with an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide.

[0005] In some embodiments, the polynucleotide sequence comprises a plurality of DRACH motifs, and the substitution is performed only on a subset of the DRACH motifs. In some embodiments, the substitution is performed only on a DRACH motif that is separated from a splice junction site within the polynucleotide sequence by at least 10 nucleotides.In some embodiments, the substitution is performed only on a DRACH motif that is separated from a splice junction site within the polynucleotide sequence by at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 180 nucleotides, at least 190 nucleotides, at least 200. Petition 870250096842, dated 10 / 23 / 2025, page 10 / 180 3 / 121 nucleotides or more.

[0006] In some embodiments, the substitution is performed only on a DRACH motif that is separated from a splice junction site within the polynucleotide sequence by about 10 to about 200 nucleotides, by about 20 to about 190 nucleotides, by about 30 to about 180 nucleotides, by about 40 to about 170 nucleotides, by about 50 to about 160 nucleotides, by about 60 to about 150 nucleotides, by about 70 to about 140 nucleotides, by about 80 to about 130 nucleotides, by about 90 to about of 120 nucleotides or by about 100 nucleotides.

[0007] In some embodiments, the substitution is performed on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, TGACT, AAACA, AAACC, AAACT, AGACC, GAACA, GGACC, TAACA, TAACC, TAACT, TGACA and TGACC. In some embodiments, the substitution is performed on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, and TGACT.

[0008] In some embodiments, the substitution of one or more nucleotides in the DRACH motif is performed using one or more codon usage guidelines selected from the group consisting of: (a) never use the GAC codon to encode aspartic acid when eliminating the DRACH motif; (b) never use the GAA codon to encode glutamic acid when eliminating the DRACH motif; (c) never use the GGA codon to encode glycine when eliminating the DRACH motif; (d) never use the AAA codon to encode lysine when eliminating the Petition 870250096842, dated 10 / 23 / 2025, page 11 / 180 4 / 121 reason DRACH; (e) never use the AAC codon to encode asparagine when eliminating the DRACH motif; and / or (f) always use the ACG codon to encode threonine when eliminating the DRACH motif.

[0009] In some embodiments, codon optimization also includes the implementation, throughout the polynucleotide sequence, of one or more codon usage guidelines selected from the group consisting of: (a) never use the GAC codon to encode aspartic acid by the full polynucleotide sequence; (b) never use the GAA codon to encode glutamic acid by the full polynucleotide sequence; (c) never use the GGA codon to encode glycine by the full polynucleotide sequence; (d) never use the AAA codon to encode lysine by the full polynucleotide sequence; (e) never use the AAC codon to encode asparagine by the full polynucleotide sequence; and / or (f) never use the ACG codon to encode threonine by the full polynucleotide sequence.

[0010] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides that together make up a synonymous codon that occurs naturally at a higher frequency in a target organism than the frequency with which the unmodified codon occurs in the target organism.

[0011] In some modalities, before the replacement of one or Petition 870250096842, dated 10 / 23 / 2025, page 12 / 180 5 / 121 more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism. In some embodiments, the target organism is a mammal, optionally where the mammal is a human.

[0012] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by means of a web-based codon optimization tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

[0013] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by modulating the guanine-cytosine (GC) content of the polynucleotide, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

[0014] In some embodiments, the GC content of the polynucleotide is adjusted to between 50% and 80%. In some embodiments, the GC content of the polynucleotide is adjusted to between 65% and 75%.

[0015] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method further comprises replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides which together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism relative to the frequency with which the unmodified codon occurs in the target organism.

[0016] In some modalities, after the replacement of one or Petition 870250096842, dated 10 / 23 / 2025, page 13 / 180 6 / 121 more nucleotides in the DRACH motif, the codon optimization method further comprises replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism. In some embodiments, the target organism is a mammal, optionally where the mammal is a human.

[0017] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method further comprises optimizing by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

[0018] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method also involves modulating the GC content, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

[0019] In some embodiments, the GC content of the polynucleotide is adjusted to between 50% and 80%. In some embodiments, the GC content of the polynucleotide is adjusted to between 65% and 75%.

[0020] In some embodiments, the amino acid sequence of the protein or polypeptide shares at least 85% sequence identity with a wild-type amino acid sequence of the protein or polypeptide after codon optimization. In some embodiments, the amino acid sequence of the protein or polypeptide shares 100% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

[0021] In some modalities, the number of DRACH motifs in Petition 870250096842, dated 10 / 23 / 2025, p. 14 / 180 7 / 121 The polynucleotide sequence is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, compared to a corresponding polynucleotide sequence that has not been subjected to the method. In some embodiments, the number of DRACH motifs in the polynucleotide sequence is reduced by at least 100% compared to a corresponding polynucleotide sequence that has not been subjected to the method.

[0022] In some embodiments, the codon optimization method results in increased stability or half-life of the mRNA transcript.

[0023] In some embodiments, the increase in stability or half-life of the mRNA transcript is assessed by an increase in concentration or relative abundance compared to a corresponding polynucleotide sequence that has not been subjected to the method.

[0024] In some embodiments, the increase in concentration or relative abundance of the mRNA transcript compared to the mRNA transcript of the native polynucleotide sequence is measured by RT-PCR, fluorimetry, colorimetry, spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses, or a matrix using one or more hybridizing oligonucleotides or antibodies against the mRNA transcript of interest.

[0025] In some embodiments, the increase in stability or half-life of the mRNA transcript is assessed by reduced decay rates, as detected by one or more pulse-chasing methodologies.

[0026] In some embodiments, reducing the number of DRACH motifs in the polynucleotide sequence reduces the number of N(6)methyladenosine (m6A) modifications in an mRNA transcript compared with a corresponding polynucleotide sequence that has not been subjected to the method.

[0027] In some modalities, the optimization method of Petition 870250096842, dated 10 / 23 / 2025, p. 15 / 180 8 / 121 codons results in increased protein expression or stability compared to the protein encoded by a corresponding polynucleotide sequence that has not been subjected to the method. In some embodiments, the corresponding polynucleotide sequence is a wild-type polynucleotide sequence.

[0028] In some embodiments, protein expression or stability is elevated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% compared to a corresponding polynucleotide sequence that has not been subjected to the method.

[0029] In some embodiments, protein expression or stability is elevated by about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times or more than 50 times compared to a corresponding polynucleotide sequence that has not been subjected to the method.

[0030] In some embodiments, the increase in protein expression is measured by mass spectrometry, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation methods, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or a matrix using aptamers or antibodies against the protein of interest.

[0031] In another aspect, the disclosure presents a polynucleotide produced by the method of any of the previous aspects or embodiments of the disclosure.

[0032] In another aspect, the disclosure presents a method for distributing a polynucleotide sequence encoding a polypeptide to a Petition 870250096842, dated 10 / 23 / 2025, page 16 / 180 9 / 121 host cell, the method comprising (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent amount of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) providing the resulting polynucleotide sequence to the host cell.

[0033] In a further aspect, the disclosure presents a method for expressing an mRNA transcript in a host cell from a polynucleotide sequence encoding a polypeptide, the method comprising (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent amount of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) providing the polynucleotide sequence to the host cell.

[0034] In yet another aspect, the disclosure presents a method for expressing a protein in a host cell from a polynucleotide sequence that encodes the protein, the method comprising (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded protein and (ii) providing the polynucleotide sequence to the host cell.

[0035] In a further aspect, the disclosure presents a method for distributing a polynucleotide sequence encoding a polypeptide to a subject, the method comprising (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide. Petition 870250096842, dated 10 / 23 / 2025, page 17 / 180 10 / 121 and (ii) provide the resulting polynucleotide sequence to the subject.

[0036] In a further aspect, the disclosure presents a method for expressing an mRNA transcript in a subject from a polynucleotide sequence encoding a polypeptide, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) providing the polynucleotide sequence to the subject.

[0037] In another aspect, the disclosure presents a method for expressing a protein in a subject from a polynucleotide sequence encoding the protein, the method comprising (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded protein and (ii) providing the polynucleotide sequence to the subject.

[0038] In some embodiments of any of the preceding aspects of the disclosure, the substitution is made in a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, TGACT, AAACA, AAACC, AAACT, AGACC, GAACA, GGACC, TAACA, TAACC, TAACT, TGACA and TGACC. In some embodiments, the substitution is made in a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, and TGACT.

[0039] In some embodiments, the substitution of one or more nucleotides in the DRACH motif is performed using one or more codon usage guidelines selected from the group consisting of: (a) never use the GAC codon to encode aspartic acid Petition 870250096842, dated 10 / 23 / 2025, page 18 / 180 11 / 121 remove the DRACH reason; (b) never use the GAA codon to encode glutamic acid when eliminating the DRACH motif; (c) never use the GGA codon to encode glycine when eliminating the DRACH motif; (d) never use the AAA codon to encode lysine when eliminating the DRACH motif; (e) never use the AAC codon to encode asparagine when eliminating the DRACH motif; and / or (f) always use the ACG codon to encode threonine when eliminating the DRACH motif. [00 40] In some embodiments, codon optimization also includes the implementation, throughout the polynucleotide sequence, of one or more codon usage guidelines selected from the group consisting of: (a) never use the GAC codon for aspartic acid by the full polynucleotide sequence; (b) never use the GAA codon for glutamic acid by the full polynucleotide sequence; (c) never use the GGA codon for glycine by the full polynucleotide sequence; (d) never use the AAA codon for lysine by the full polynucleotide sequence; (e) never use the AAC codon for asparagine by the full polynucleotide sequence; and / or (f) never use the ACG codon for threonine by the full polynucleotide sequence.

[0041] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was optimized Petition 870250096842, dated 10 / 23 / 2025, p. 19 / 180 12 / 121 in codon by replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides that together make up a synonymous codon that occurs naturally at a higher frequency in a target organism than the frequency with which the unmodified codon occurs in the target organism.

[0042] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism. In some embodiments, the target organism is a mammal, optionally where the mammal is a human.

[0043] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by means of a web-based codon optimization tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

[0044] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was optimized per codon by modulating the polynucleotide GC content, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

[0045] In some embodiments, the GC content of the polynucleotide is adjusted to between 50% and 80%. In some embodiments, the GC content of the polynucleotide is adjusted to between 65% and 75%.

[0046] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises substituting a Petition 870250096842, dated 10 / 23 / 2025, page 20 / 180 13 / 121 or more nucleotides in a codon are replaced by an equivalent number of alternative nucleotides that together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism than the frequency with which the unmodified codon occurs in the target organism.

[0047] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism. In some embodiments, the target organism is a mammal, optionally where the mammal is a human.

[0048] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises optimization by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

[0049] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method also involves modulating the GC content, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

[0050] In some embodiments, the GC content of the polynucleotide is adjusted to between 50% and 80%. In some embodiments, the GC content of the polynucleotide is adjusted to between 65% and 75%.

[0051] In some embodiments, the amino acid sequence of the protein or polypeptide shares at least 85% identity with Petition 870250096842, dated 10 / 23 / 2025, p. 21 / 180 14 / 121 sequence with a wild-type amino acid sequence of the protein or polypeptide after codon optimization.

[0052] In some embodiments, the amino acid sequence of the protein or polypeptide shares 100% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

[0053] In some embodiments, the number of DRACH motifs in the polynucleotide sequence is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, compared with a corresponding polynucleotide sequence that has not been subjected to the method.

[0054] In some embodiments, the number of DRACH motifs in the polynucleotide sequence is reduced by at least 100% compared with a corresponding polynucleotide sequence that has not been subjected to the method.

[0055] In some embodiments, the method results in increased stability or half-life of the mRNA transcript.

[0056] In some embodiments, the increase in stability or half-life of the mRNA transcript is assessed by an increase in concentration or relative abundance compared to a corresponding polynucleotide sequence that has not been subjected to the method.

[0057] In some embodiments, the increase in concentration or relative abundance of the mRNA transcript compared to the mRNA transcript of the native polynucleotide sequence is measured by RT-PCR, fluorimetry, colorimetry, spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses, or a matrix using one or more hybridizing oligonucleotides or antibodies against the mRNA transcript of interest. Petition 870250096842, dated 10 / 23 / 2025, page 22 / 180 15 / 121

[0058] In some embodiments, the increase in mRNA transcript stability or half-life is assessed by reduced decay rates, as detected by one or more pulse-chasing methodologies.

[0059] In some embodiments, reducing the number of DRACH motifs in the polynucleotide sequence reduces the number of N(6)methyladenosine (m6A) modifications in an mRNA transcript compared with a corresponding polynucleotide sequence that has not been subjected to the method.

[0060] In some embodiments, the method results in increased expression or stability of the protein compared to the protein encoded by a corresponding polynucleotide sequence that was not subjected to the method.

[0061] In some embodiments, the corresponding polynucleotide sequence is a wild-type polynucleotide sequence.

[0062] In some embodiments, protein expression or stability is elevated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% compared to a corresponding polynucleotide sequence that has not been subjected to the method.

[0063] In some embodiments, protein expression or stability is elevated by about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times or more than 50 times compared to a corresponding polynucleotide sequence that has not been subjected to the method.

[0064] In some modalities, the increase in protein expression is measured by mass spectrometry, Western blot analysis, assay Petition 870250096842, dated 10 / 23 / 2025, page 23 / 180 16 / 121 enzyme-linked immunosorbent assay (ELISA), immunoprecipitation methods, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or a matrix using aptamers or antibodies against the protein of interest.

[0065] In some embodiments, the polynucleotide is delivered to the host cell through contact of the host cell with a vehicle comprising the polynucleotide.

[0066] In some embodiments, the polynucleotide is delivered to the subject by administering to the subject a vehicle comprising the polynucleotide.

[0067] In some embodiments, the vehicle is selected from the group consisting of a lipid nanoparticle, a liposome, a viral genome, and a viral vector.

[0068] In some forms, the viral vector is an adeno-associated virus (AAV), an adenovirus, a retrovirus, or a lentivirus.

[0069] In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV is pseudotyped. In some embodiments, the AAV comprises an inverted terminal repeat (ITR) sequence and a capsid protein derived from different serotypes.

[0070] In some forms, delivery to the host cell occurs in vivo, in vitro or ex vivo.

[0071] In some embodiments, administration to the subject occurs by means of (i) in vivo administration of the polynucleotide to the subject or (ii) ex vivo expression of the polynucleotide in a host cell, followed by administration of the host cell to the subject.

[0072] In some modalities, the method is used to treat a subject who has or is at risk of having a disease characterized by low expression or activity of a protein.

[0073] In another aspect, the disclosure presents a method for treating a subject who has or is at risk of developing a disease, the method Petition 870250096842, dated 10 / 23 / 2025, p. 24 / 180 17 / 121 comprising (i) replacing one or more nucleotides in a DRACH motif within a polynucleotide sequence encoding a disease-associated polynucleotide with an equivalent amount of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) administering the polynucleotide sequence to the subject.

[0074] In a further aspect, the disclosure presents a method for treating a subject who has or is at risk of developing a disease, the method comprising administering a polynucleotide sequence encoding a disease-associated polypeptide to the subject, wherein prior to administration, one or more nucleotides in a DRACH motif within the polynucleotide sequence have been replaced by an equivalent amount of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide.

[0075] In some embodiments, the substitution is performed on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, TGACT, AAACA, AAACC, AAACT, AGACC, GAACA, GGACC, TAACA, TAACC, TAACT, TGACA and TGACC. In some embodiments, the substitution is performed on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, and TGACT.

[0076] In some embodiments, the substitution of one or more nucleotides in the DRACH motif is performed using one or more codon usage guidelines selected from the group consisting of: (a) never use the GAC codon to encode aspartic acid when eliminating the DRACH motif; (b) never use the GAA codon to encode glutamic acid when eliminating the DRACH motif; Petition 870250096842, dated 10 / 23 / 2025, page 25 / 180 18 / 121 (c) never use the GGA codon to encode glycine when deleting the DRACH motif; (d) never use the AAA codon to encode lysine when eliminating the DRACH motif; (e) never use the AAC codon to encode asparagine when eliminating the DRACH motif; and / or (f) always use the ACG codon to encode threonine when eliminating the DRACH motif. [00 77] In some embodiments, prior to administration, the polynucleotide sequence has been codon-optimized by a method comprising implementing, across the total polynucleotide sequence, one or more codon usage guidelines selected from the group consisting of: (a) never use the GAC codon to encode aspartic acid by the full polynucleotide sequence; (b) never use the GAA codon to encode glutamic acid by the full polynucleotide sequence; (c) never use the GGA codon to encode glycine by the full polynucleotide sequence; (d) never use the AAA codon to encode lysine by the full polynucleotide sequence; (e) never use the AAC codon to encode asparagine by the full polynucleotide sequence; and / or (f) never use the ACG codon to encode threonine by the full polynucleotide sequence.

[0078] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides that together make up a Petition 870250096842, dated 10 / 23 / 2025, page 26 / 180 19 / 121 is a synonymous codon that occurs naturally at a higher frequency in a target organism compared to the frequency with which the unmodified codon occurs in the target organism.

[0079] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism. In some embodiments, the target organism is a mammal, optionally where the mammal is a human.

[0080] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by means of a web-based codon optimization tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

[0081] In some embodiments, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by modulating the guanine-cytosine (GC) content of the polynucleotide, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

[0082] In some embodiments, the GC content of the polynucleotide is adjusted to between 50% and 80%. In some embodiments, the GC content of the polynucleotide is adjusted to between 65% and 75%.

[0083] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides which together comprise a synonymous codon that Petition 870250096842, dated 10 / 23 / 2025, page 27 / 180 20 / 121 occurs naturally at a higher frequency in a target organism compared to the frequency with which the unmodified codon occurs in the target organism.

[0084] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism. In some embodiments, the target organism is a mammal, optionally where the mammal is a human.

[0085] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises optimization by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

[0086] In some embodiments, after the substitution of one or more nucleotides in the DRACH motif, the method also involves modulating the GC content, secondary polynucleotide structure, mRNA motifs, ribosomal binding sites and / or translation rates.

[0087] In some embodiments, the GC content of the polynucleotide is adjusted to between 50% and 80%. In some embodiments, the GC content of the polynucleotide is adjusted to between 65% and 75%.

[0088] In some embodiments, the amino acid sequence of the protein or polypeptide shares at least 85% sequence identity with a wild-type amino acid sequence of the protein or polypeptide after codon optimization.

[0089] In some forms, the amino acid sequence of Petition 870250096842, dated 10 / 23 / 2025, p. 28 / 180 21 / 121 protein or polypeptide shares 100% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

[0090] In some embodiments, the number of DRACH motifs in the polynucleotide sequence is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, compared with a corresponding polynucleotide sequence that has not been subjected to the method.

[0091] In some embodiments, the number of DRACH motifs in the polynucleotide sequence is reduced by at least 100% compared with a corresponding polynucleotide sequence that has not been subjected to the method.

[0092] In some embodiments, the method results in increased stability or half-life of the mRNA transcript.

[0093] In some embodiments, the high stability of the mRNA transcript or half-life is assessed by an elevation in concentration or relative abundance compared to a reference.

[0094] In some embodiments, the increase in concentration or relative abundance of the mRNA transcript compared to the mRNA transcript of the native polynucleotide sequence is measured by RT-PCR, fluorimetry, colorimetry, spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses, or a matrix using one or more hybridizing oligonucleotides or antibodies against the mRNA transcript of interest.

[0095] In some embodiments, reducing the number of DRACH motifs in the polynucleotide sequence reduces the number of N(6)methyladenosine (m6A) modifications in an mRNA transcript compared with a corresponding polynucleotide sequence that has not been subjected to the method. Petition 870250096842, dated 10 / 23 / 2025, page 29 / 180 22 / 121

[0096] In some modalities, the method results in increased expression or stability of the protein compared to a reference.

[0097] In some embodiments, the corresponding polynucleotide sequence is a wild-type polynucleotide sequence.

[0098] In some modalities, the reference is a sample obtained from a subject who has not received treatment or a sample obtained from the subject before treatment.

[0099] In some embodiments, protein expression or stability is elevated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% compared to a reference.

[0100] In some modalities, protein expression or stability is elevated by approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 25 times, approximately 30 times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times or more than 50 times compared to a reference.

[0101] In some embodiments, the increase in protein expression is measured by mass spectrometry, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation methods, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses or a matrix using aptamers or antibodies against the protein of interest.

[0102] In some embodiments, the polynucleotide is administered to the subject by contacting the subject with a vehicle comprising the polynucleotide.

[0103] In some embodiments, the vehicle is selected from a lipid nanoparticle, a liposome, a viral genome, and a viral vector. In Petition 870250096842, dated 10 / 23 / 2025, page 30 / 180 23 / 121 In some embodiments, the viral vector is an adeno-associated virus (AAV), an adenovirus, a retrovirus, a lentivirus, or a double-stranded DNA virus. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV is pseudotyped. In some embodiments, the AAV comprises an inverted terminal repeat (ITR) sequence and a capsid protein derived from different serotypes.

[0104] In some modalities, administration occurs by directly supplying the subject, in vivo, with the polynucleotide or by expressing the polynucleotide in a host cell ex vivo and subsequently administering the host cell to the subject.

[0105] In some forms, administration is performed via intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intraarterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous routes.

[0106] In some modalities, treatment is monotherapy. In some modalities, the method is combined with the administration of one or more additional therapeutic agents.

[0107] In a further aspect, the disclosure presents a kit comprising a package insert instructing a user to perform a codon optimization method comprising modifying a polynucleotide sequence encoding a polypeptide by replacing one or more nucleotides with a DRACH motif in the polynucleotide sequence without altering the amino acid sequence of the encoded polypeptide, thereby eliminating one or more DRACH motifs.

[0108] In a further aspect, the disclosure presents a kit comprising a leaflet instructing a user on how to perform the method of any of the aspects or modalities above the disclosure.

[0109] In some embodiments, the kit comprises a device for delivering the polynucleotide sequence to a subject. In some Petition 870250096842, dated 10 / 23 / 2025, p. 31 / 180 In the 24 / 121 modes, the kit comprises one or more binding molecules to detect the expression of an mRNA transcript encoding the polypeptide or the expression or activity of the polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] The attached drawings are included to illustrate ways of carrying out the disclosure and to promote a further understanding of its implementations.

[0111] FIG. 1 is a diagram showing the map features of the pAAV ZsGreen1 plasmid.

[0112] FIG. 2A is a bar graph showing the relative production of the fluorescent protein ZsGreen from all best codon-optimized open reading frames (ORFs) with or without DRACH motif removal. Flow cytometry measurements of HEK293T cells were performed 2 or 3 days after a 1:4 transfection with plasmids containing the best codon-optimized ORFs, in which all codons were replaced with synonymous codons with the highest frequency of use according to Table 1. “+m6A” denotes that 14 DRACH motifs were present in the transfected ORF, and sample groups labeled “-m6A” were further modified with the six codon usage rules to eliminate all DRACH motifs present in the coding sequence before transfection.

[0113] FIG. 2B is a bar graph showing the relative production of the ZsGreen fluorescent protein of all the best codon-optimized ORFs with or without removal of DRACH motifs. Flow cytometry measurements of HEK293T cells were performed 2 days after a 1:16 transfection with plasmids containing the best codon-optimized ORFs, in which all codons were replaced by synonymous codons with the highest frequency of use according to Table 1. “+m6A” denotes that 14 DRACH motifs were present in the transfected ORF, and “-m6A” denotes Petition 870250096842, dated 10 / 23 / 2025, p. 32 / 180 25 / 121 that the ORFs were further modified with the six codon usage rules to eliminate all DRACH motifs present in the coding sequence before transfection.

[0114] FIG. 3A is a bar graph showing the relative production of the ZsGreen fluorescent protein from proportional use-codon optimized ORFs with or without DRACH motif removal. Flow cytometry measurements of HEK293T cells were performed 2 or 3 days after a 1:4 transfection with plasmids containing the best proportional use-codon optimized ORFs, in which all codons were represented based on their reported frequency in Table 1. “+m6A” denotes that 16 DRACH motifs were present in the transfected ORF and “-m6A” denotes that the ORFs were further modified with the six codon usage rules to eliminate all DRACH motifs present in the coding sequence before transfection.

[0115] FIG. 3B is a bar graph showing the relative production of the fluorescent protein ZsGreen from all best open reading frames (ORFs) optimized by codons with or without removal of DRACH motifs. Flow cytometry measurements of HEK293T cells were performed 2 days after a 1:16 transfection with plasmids containing the best codon-optimized ORFs of proportional use, in which all codons were represented based on their reported frequency in Table 1. “+m6A” denotes that 16 DRACH motifs were present in the transfected ORF and “-m6A” denotes that the ORFs were further modified with the six codon usage rules to eliminate all DRACH motifs present in the coding sequence before transfection.

[0116] FIG. 4 is a bar graph showing the relative secretion of GLP-1-Fc protein from ORFs that were codon-optimized using a commercially available codon optimization web tool, with Petition 870250096842, dated 10 / 23 / 2025, page 33 / 180 26 / 121 or without further modifications to remove DRACH motifs. ORFs in which DRACH motifs have been removed are denoted as “-m6A”. Protein secretion was measured via enzyme-linked immunosorbent assay (ELISA).

[0117] FIG. 5 is a bar graph showing the relative secretion of GLP-1-Fc protein measured by HEK293T cells transfected with AAVs containing ORFs that were codon-optimized using four distinct strategies. “CH” denotes that the GLP-1-Fc ORF was codon-optimized by replacing each codon with a synonymous codon with higher usage. “IDT” denotes that the GLP-1-Fc ORF was codon-optimized using a commercially available codon optimization web tool. “-m6A” further denotes that, following the indicated codon optimization strategy, the ORFs were modified to remove DRACH sequences. Protein concentrations were measured by ELISA.

[0118] FIG. 6A is a graph showing detectable levels of GLP-1-Fc protein in the serum of mice on different days after a low-dose intramuscular administration (1 x 10⁹ genome copies per mouse) of AAVs containing the native ORF or an ORF that was codon-optimized using one of four distinct strategies. “CH” denotes that the GLP-1-Fc ORF was codon-optimized, replacing each codon with a synonymous codon with higher usage. “IDT” denotes that the GLP-1-Fc ORF was codon-optimized using a commercially available codon optimization web tool. “m6A” denotes that, following the indicated codon optimization strategy, the ORFs were modified to remove DRACH sequences. Protein concentrations were measured by ELISA.

[0119] FIG. 6B is a graph showing detectable levels of GLP-1-Fc protein in the serum of mice on different days after a high-dose intramuscular administration (1 x 1010 genome copies per Petition 870250096842, dated 10 / 23 / 2025, p. 34 / 180 27 / 121 mouse) of AAVs containing the native ORF or an ORF that was codon-optimized using one of four distinct strategies. “CH” denotes that the GLP-1-Fc ORF was codon-optimized, replacing each codon with a synonymous codon with higher usage. “IDT” denotes that the GLP-1-Fc ORF was codon-optimized using a commercially available codon optimization web tool. “m6A” also denotes that, following the indicated codon optimization strategy, the ORFs were modified to remove DRACH sequences. Protein concentrations were measured by ELISA.

[0120] FIG. 7 is a bar graph showing detectable levels of GLP-1-Fc protein in the serum of mice 14 days after receiving a low-dose intramuscular administration (1 x 109 genome copies per mouse) of AAVs containing either a native ORF or an ORF that was codon-optimized using one of two distinct codon optimization strategies. “CH” denotes that the GLP-1-Fc ORF was codon-optimized, replacing each codon with a synonymous codon with the highest usage. “m6A” denotes that, after codon optimization, the ORFs were further modified to remove DRACH sequences. Protein concentrations were measured by ELISA.

[0121] FIG. 8A is a bar graph showing detectable levels of GLP-1-Fc protein in the serum of mice 28 days after receiving a low-dose intramuscular administration (1 x 109 genome copies per mouse) of AAVs containing ORFs that were codon-optimized using a commercially available codon optimization web tool in which DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). Protein concentrations were measured by ELISA.

[0122] FIG. 8B is a bar graph showing detectable levels of GLP-1-Fc protein in the serum of mice 28 days after receiving a low-dose intramuscular administration (1 x 109 copies of Petition 870250096842, dated 10 / 23 / 2025, page 35 / 180 28 / 121 genome per mouse) of AAVs containing ORFs that were codon-optimized by replacing each codon with a synonymous codon having the highest usage in which the DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). Protein concentrations were measured by ELISA.

[0123] FIG. 8C is a bar graph showing detectable levels of GLP-1-Fc protein in the serum of mice 28 days after receiving a high-dose intramuscular administration (1 x 1010 genome copies per mouse) of AAVs containing ORFs that were codon-optimized using a commercially available codon optimization web tool in which DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). Protein concentrations were measured by ELISA.

[0124] FIG. 8D is a bar graph showing detectable levels of GLP-1-Fc protein in the serum of mice 28 days after receiving a high-dose intramuscular administration (1 x 1010 genome copies per mouse) of AAVs containing ORFs that were codon-optimized by replacing each codon with a synonymous codon having the highest usage in which the DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). Protein concentrations were measured by ELISA.

[0125] FIG. 9A is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in the muscle tissue of mice 60 days after receiving a low-dose intramuscular administration (1 x 109 genome copies per mouse) of AAVs containing ORFs that were codon-optimized using a commercially available web-based codon optimization tool in which DRACH motifs were left unchanged (+m6A) or removed by additional substitution (-m6A). The Petition 870250096842, dated 10 / 23 / 2025, page 36 / 180 29 / 121 transcription levels were measured by qPCR.

[0126] FIG. 9B is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in the muscle tissue of mice 60 days after receiving a low-dose intramuscular administration (1 x 109 genome copies per mouse) of AAVs containing ORFs that were codon-optimized by replacing each codon with a synonymous codon with the highest usage, in which DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). Transcription levels were measured by qPCR.

[0127] FIG. 9C is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in mouse muscle tissue 60 days after receiving a high-dose intramuscular administration (1 x 1010 genome copies per mouse) of AAVs containing ORFs that were codon-optimized using a commercially available web-based codon optimization tool in which DRACH motifs were left unchanged (+m6A) or removed by additional substitution (-m6A). Transcription levels were measured by qPCR.

[0128] FIG. 9D is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in the muscle tissue of mice 60 days after receiving a high-dose intramuscular administration (1 x 1010 genome copies per mouse) of AAVs containing ORFs that were codon-optimized by replacing each codon with a synonymous codon with the highest usage, in which DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). Transcription levels were measured by qPCR.

[0129] FIG. 10A is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in the liver tissue of mice 60 days after receiving a low intramuscular administration of Petition 870250096842, dated 10 / 23 / 2025, page 37 / 180 30 / 121 doses (1 x 109 genome copies per mouse) of AAVs containing ORFs that were codon-optimized using a commercially available web-based codon optimization tool in which DRACH motifs were left unchanged (+m6A) or removed by additional substitution (-m6A). Transcription levels were measured by qPCR.

[0130] FIG. 10B is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in the liver tissue of mice 60 days after receiving a low-dose intramuscular administration (1 x 109 genome copies per mouse) of AAVs containing ORFs that were codon-optimized by replacing each codon with a synonymous codon with the highest usage, in which DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). Transcription levels were measured by qPCR.

[0131] FIG. 10C is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in the liver tissue of mice 60 days after receiving a high-dose intramuscular administration (1 x 1010 genome copies per mouse) of AAVs containing ORFs that were codon-optimized using a commercially available web-based codon optimization tool in which DRACH motifs were left unchanged (+m6A) or removed by additional substitution (-m6A). Transcription levels were measured by qPCR.

[0132] FIG. 10D is a bar graph showing the relative levels of GLP-1-Fc mRNA transcription in the liver tissue of mice 60 days after receiving a high-dose intramuscular administration (1 x 1010 genome copies per mouse) of AAVs containing ORFs that were codon-optimized by replacing each codon with a synonymous codon with the highest usage, in which the DRACH motifs were left unchanged (+m6A) or were removed by additional substitution (-m6A). The Petition 870250096842, dated 10 / 23 / 2025, page 38 / 180 31 / 121 transcription levels were measured by qPCR. DEFINITIONS

[0133] Unless otherwise defined in this document, the scientific and technical terms used in this document have the meanings that are commonly understood by those skilled in the art. In case of any latent ambiguity, the definitions provided in this document take precedence over any dictionary or extrinsic definition. Unless otherwise required by the context, singular terms should include plurals and plural terms should include singulars. The use of or means and / or, unless otherwise indicated. The use of the term including, as well as other forms such as includes and included, is not limiting.

[0134] As used herein, the term about, when applied to one or more values ​​of interest, refers to a value that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in any direction (greater or less than) of a stated reference value, unless otherwise indicated or evident from the context (except where such number exceeds 100% of a possible value).

[0135] As used herein, the term “codon optimization” refers to a process of altering codons in a given gene or coding sequence in such a way as to increase the expression of the encoded polypeptide without altering the amino acid sequence of the polypeptide. This process utilizes the redundancy of the genetic code, in which multiple combinations of three-base-pair codons code for a single amino acid. These codons, which differ in nucleic acid sequence but code for the same amino acid, are here referred to as “synonymous codons.” As a non-limiting example, the amino acid leucine can be coded by any of the following six codons: UUA, UUG, CUA, CUG, CUU, or CUC, all of which are synonymous with each other. The codon optimization process generally Petition 870250096842, dated 10 / 23 / 2025, page 39 / 180 32 / 121 improves mRNA expression efficiency, mRNA stability, and / or polypeptide synthesis compared to the unmodified gene or coding sequence. Codon optimization generally improves the expression of a target gene by modifying a codon sequence in a way that takes into account the abundance of tRNA in a cell type or organism, secondary structural elements in the polynucleotide sequence, and / or binding interactions of the polynucleotide sequence and one or more binding partners (e.g., ribosomes, methyltransferases, tRNA molecules, and other binding partners known in the art). An “alternative nucleotide” or “alternative codon” in reference to codon optimization refers to a different nucleotide or codon that ultimately codes for the same amino acid sequence of a polypeptide or protein of interest.

[0136] As used herein, the terms “codon usage table”, “codon bias table”, “codon frequency lookup table”, and the like are used interchangeably to refer to a table that correlates each codon that can be used to encode a specific amino acid with the frequency with which that codon is used to encode its corresponding amino acid in a specific setting. The setting might be, for example, (i) a specific organism (e.g., a mammalian subject, such as a human), (ii) a specific class of genes within a given organism, or (iii) one or more synthetic polynucleotides. A hybrid codon usage table or hybrid codon bias table may also be constructed by combining two or more codon usage tables according to a variety of possible rules.

[0137] As used herein, an “organism” or a “target organism,” in reference to codon optimization, refers to the organism intended to receive the delivery of one or more polynucleotides (e.g., one or more codon-optimized polynucleotides) described herein. Petition 870250096842, dated 10 / 23 / 2025, p. 40 / 180 33 / 121

[0138] As used herein, a “coding sequence” refers to an open reading frame (ORF) in a nucleic acid that, upon expression, yields a polypeptide or protein. An ORF is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG in the context of a DNA sequence or AUG in the context of an RNA sequence)) and ends with a stop codon (e.g., TAA, TAG, or TGA in the context of a DNA sequence, or UAA, UAG, or UGA in the context of an RNA sequence). An ORF typically encodes a protein. It is understood that the sequences disclosed herein may include further elements, e.g., 5' and 3' untranslated regions (UTRs), but that these elements, unlike the ORF, do not necessarily need to be present in an RNA polynucleotide (e.g., an mRNA transcript) disclosed herein.

[0139] As used herein in the context of a target protein product, the terms “expression level” or “expression level” are used interchangeably and refer to the amount of the protein product in a subject or biological sample. “Expression” of a desired protein, as the term is used herein, refers to the general process by which a gene of interest is processed (e.g., in a subject, in a cell, or ex vivo by means of one or more enzymes or organelles obtained from a cell) so as to produce the protein product. Specifically, the term “expression” may be used to describe one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.Consequently, the genes that are expressed using the compositions and methods described here include those that are... Petition 870250096842, dated 10 / 23 / 2025, page 41 / 180 34 / 121 transcribed into a polynucleotide (such as mRNA) and then translated into a polypeptide or protein, as well as those genes that are transcribed into an RNA polynucleotide but are not translated into a polypeptide (e.g., transfer and ribosomal RNAs). Fragments of the transcribed polynucleotide, the translated polypeptide, or modifications of the polynucleotide and / or polypeptide (e.g., post-translational modification of a polypeptide) should also be considered expressed, regardless of whether they originate from a transcript generated by alternative splicing or from a degraded transcript, or from post-translational processing of the polypeptide, e.g., by proteolysis.

[0140] The expression of a gene of interest in a subject may be manifested, for example, by the detection of: an increase in the amount or concentration of mRNA encoding a corresponding protein (as assessed, for example, using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR), RT-PCR and RNA sequencing techniques), an increase in the amount or concentration of a corresponding protein (as assessed, for example, using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), mass spectrometry, Western blot analysis, flow cytometry, immunofluorescence, colorimetry assays or a matrix using targeting antibodies or hybridization nucleotides, among others) and / or an increase in the activity of a corresponding protein (for example, in the case of an enzyme,as assessed using an enzyme activity assay known in the art) in a sample obtained from the subject.

[0141] As used herein, the terms “polynucleotide” or “nucleic acid” refer to polymers of nucleotides of any length and include DNA and / or RNA. Nucleotides can be deoxyribonucleotides, Petition 870250096842, dated 10 / 23 / 2025, page 42 / 180 35 / 121 ribonucleotides, modified nucleotides (e.g., containing modified nucleobases) and / or a nucleotide analog that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Thus, for example, polynucleotides as defined herein include, without limitation, single-stranded and double-stranded DNA, DNA including single-stranded and double-stranded regions, single-stranded and double-stranded RNA and RNA including single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA which may be single-stranded or, more typically, double-stranded or include single-stranded and double-stranded regions. In addition, the term “polynucleotide,” as used herein, refers to triple-stranded regions comprising RNA or DNA or RNA and DNA. The strands in these regions may be of the same molecule or of different molecules. The regions may include all one or more molecules, but usually involve only a region of some of the molecules.One of the molecules in a triple helix region is usually an oligonucleotide.

[0142] The term “polynucleotide” specifically includes complementary DNA (cDNA). In some embodiments, a polynucleotide is a codon-optimized gene product, in which a polynucleotide (e.g., a polynucleotide encoding a protein of interest) is subjected to one or more codon optimization methods described herein.

[0143] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogues. If present, the modification in the nucleotide structure may be conferred before or after the assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may also be modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, caps, substitution of one or more naturally occurring nucleotides by an analogue, modifications of internucleosides, such as those with uncharged linkages. Petition 870250096842, dated 10 / 23 / 2025, page 43 / 180 36 / 121 (for example, methyl phosphonates, phosphotriesters, phosphoamidates, carbamates and the like) and with charged linkages (for example, phosphorothioates, phosphorodithioates and the like), those containing pendant moieties, such as, for example, proteins (for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine and the like), those with intercalators (for example, acridine, psoralen and the like), those containing chelating agents (for example, metals, radioactive metals, boron, oxidative metals and the like), those containing alkylators, those with modified linkages (for example, alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s).Furthermore, any of the hydroxyl groups normally present in sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or it can be conjugated to solid or semi-solid supports. The terminal 5' and 3' OHs can be phosphorylated or replaced by amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized into standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- or 2'-azidoribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lycoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR', CO, or CH2 (formacetal), where each R or R' is independently H or alkyl substituted or unsubstituted (1-20 C). Petition 870250096842, dated 10 / 23 / 2025, p. 44 / 180 37 / 121 optionally containing an ether (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0144] As used herein, the terms “adenine” and “adenosine” are interchangeable terms with reference to a nucleotide having an adenine base. As used herein, the terms “cytosine” and “cytidine” are interchangeable terms with reference to a nucleotide having a cytosine base. As used herein, the terms “guanine” and “guanidine” are interchangeable terms with reference to a nucleotide having a guanine base. As used herein, the terms “thymine” and “thymidine” are interchangeable terms with reference to a nucleotide having a thymine base. As used herein, the terms “uracil” and “uridine” are interchangeable terms with reference to a nucleotide having a uracil base.

[0145] As used herein, the term “splice junction site” refers to a region in a nucleic acid sequence (e.g., DNA or RNA) that is the boundary between an intron and an exon in a gene. Characteristics of splice junction sites or “splice locations,” as well as splice mechanisms, are described in the art, such as Roca et al. Genes Dev. 27(2):129-144, 2013, which is incorporated herein by reference.

[0146] As used herein, “messenger RNA”, “mRNA” or “mRNA transcript” is any RNA molecule that encodes one (at least one) polypeptide or fragment thereof and can be translated to produce the encoded polypeptide or fragment thereof in vitro, in vivo, in situ or ex vivo. Structural and topological features, as well as posttranscriptional modifications of mRNA, are described herein and are well known in the art.

[0147] As used herein, the term “peptide” refers to a polymer containing a plurality of amino acid monomers (or analogues) Petition 870250096842, dated 10 / 23 / 2025, page 45 / 180 38 / 121 of the same) and that is less than or equal to 50 amino acids in length (for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length).

[0148] As used herein, the term “polypeptide” refers to a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, encompasses polypeptides of any size, structure, or function. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or it may be a multi-molecular complex, such as a dimer, a trimer, or a tetramer. The term polypeptide may also be applied to polymers of amino acids in which one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid.

[0149] As used herein, the term “protein” refers to contiguous amino acids or amino acid residues. Typically, proteins have a function. However, proteins also encompass polypeptides and smaller contiguous amino acid sequences that have no functional activity. Examples of functional proteins include, but are not limited to, enzymes (such as dehydrogenases, hydrolases, oxidoreductases, transferases, lyases, and ligases, among others), receptors, receptor ligands, cytokines, antibodies, immunomodulatory molecules, signaling molecules, or proteins that are labeled or modified (e.g., for diagnostic or other clinical applications). Useful general classes of enzymes include, but are not limited to, proteases, cellulases, lipases, hemicellulases, laccases, amylases, glucoamylases, esterases, lactases, polygalacturonases, galactosidases, ligninases, oxidases, peroxidases, glucose isomerases, nitrilases, hydroxylases, polymerases, and depolymerases.In addition to enzymes, the encoded proteins can also be... Petition 870250096842, dated 10 / 23 / 2025, page 46 / 180 39 / 121 used in this invention include, but are not limited to, transcription factors, antibodies, receptors, growth factors (any of the PDGFs, EGFs, FGFs, SCF, HGF, TGFs, TNFs, insulin, IGFs, LIFs, oncostatins and CSFs), immunomodulators, peptide hormones, cytokines, integrins, interleukins, adhesion molecules, thrombomodulatory molecules, protease inhibitors, angiostatins, defensins, cluster of differentiation antigens, interferons, chemokines, antigens including those of viruses and infectious organisms, oncogenic products, thrombopoietin, erythropoietin, tissue plasminogen activator and any other biologically active protein that is desired for use in a clinical setting.Also included are deletion mutants of these proteins, individual domains of these proteins, fusion proteins made from these proteins, and mixtures of these proteins; particularly useful are those that have increased half-lives and / or increased activity.

[0150] As used herein, the term “DRACH motif” refers to a short five-base-pair nucleic acid consensus sequence, in which the “D” in the DRACH consensus sequence designates guanine, adenine, or uracil; “R” designates guanine or adenine; “A” designates adenine; “C” designates cytosine; and “H” designates adenine, uracil, or cytosine. A DRACH motif may be present in a polynucleotide sequence, such as an RNA molecule (e.g., an mRNA transcript). As described herein, the presence of one or more DRACH motifs in a polynucleotide sequence may reduce the half-life of a polynucleotide (e.g., an mRNA transcript) and, in turn, reduce the concentration of a translated protein or polypeptide encoded by the polynucleotide.Without being tied to any specific theory, a DRACH motif may be more likely to be modified by an N(6)-methyladenosine (“m6A”) modification to the adenine base at the third position of the motif. Such modifications may reduce. Petition 870250096842, dated 10 / 23 / 2025, page 47 / 180 40 / 121 is the half-life of the modified mRNA transcript in a cell or sample containing the mRNA transcript. One or more DRACH motifs in a nucleic acid sequence (e.g., a polynucleotide sequence; e.g., an mRNA sequence) can be removed or eliminated by substituting one or more nucleotides in one or more DRACH motifs so as to provide a synonymous codon that replaces a codon that was part of the DRACH motif, while preserving the amino acid sequence of the encoded polypeptide product. The terms “removed” or “eliminated” and their variations in reference to a DRACH motif are understood to be interchangeable.

[0151] As used herein, the term “antibody” refers to a molecule that specifically binds to, or is immunologically reactive to, a particular antigen and includes at least the variable domain of a heavy chain, and typically includes at least the variable domains of a heavy chain and a light chain of an immunoglobulin. Antibodies and antigen-binding fragments, variants or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a Vl or Vh domain, fragments produced by a Fab expression library and anti-idiotypic antibodies (anti-Id).Antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of the immunoglobulin molecule. Furthermore, unless otherwise indicated, the term monoclonal antibody (mAb) is intended to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) that are capable of specifically binding to a target protein. The Fab and F(ab')2 fragments lack the... Petition 870250096842, dated 10 / 23 / 2025, p. 48 / 180 41 / 121 Fc fragment of an intact antibody.

[0152] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an immunoglobulin that retain the ability to bind specifically to a target antigen. The antigen-binding function of an immunoglobulin can be performed by fragments of a full-length antibody. Antibody fragments can be a Fab, F(ab')2, scFv, SMIP, diabody, a triabody, an afibody, a nanobody, an aptamer, or a domain antibody.Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the Vl, Vh, Cl, and Ch1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the Vh and Ch1 domains; (iv) an Fv fragment consisting of the Vl and Vh domains of a single arm of an antibody; (v) a dAb (Ward et al., Nature 341:544-546, 1989) including Vh and Vl domains; (vi) a dAb fragment consisting of a Vh domain; (vii) a dAb consisting of a Vh or Vl domain; (viii) an isolated complementarity-determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic binder.Furthermore, although the two domains of the Fv fragment, Vl and Vh, are encoded by separate genes, they can be joined, using recombinant methods, by a linker that allows them to be made as a single protein chain in which the Vl and Vh regions pair up to form monovalent molecules (known as single-chain Fv (scFv)). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be researched for usefulness in the same way as intact antibodies. Antigen-binding fragments can be produced by... Petition 870250096842, dated 10 / 23 / 2025, page 49 / 180 42 / 121 recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins or, in certain cases, by chemical peptide synthesis procedures known in the art.

[0153] As used herein, the term “primer” refers to an oligonucleotide, natural or synthetic, that is capable of forming a duplex with a polynucleotide template and then acting as an initiation point for nucleic acid synthesis by extending its 3' end along the template nucleic acid so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers are generally extended by a DNA polymerase. Primers typically have a length in the range of 3 to 36 nucleotides, 5 to 24 nucleotides, or 14 to 36 nucleotides. In certain respects, primers are either universal primers or non-universal primers. Primer pairs may flank a sequence of interest or a set of sequences of interest. Primers and probes may be sequence-degenerate.In certain aspects, primers bind adjacently to the target sequence, whether it is the sequence to be captured for analysis or a tag to be copied.

[0154] As used herein, the term “vector” includes a nucleic acid, for example, DNA (such as a plasmid) or RNA, containing a polynucleotide encoding a gene product of interest, optionally in combination with one or more additional elements that facilitate the expression of the gene product (such as a promoter, enhancer, untranslated region, or splicing modulator, among others). Vectors include both viral and nonviral vectors. A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a eukaryotic or prokaryotic cell. Examples of such expression vectors are disclosed in, for example, WO1994 / 011026; the disclosure of which is incorporated herein. Petition 870250096842, dated 10 / 23 / 2025, page 50 / 180 43 / 121 in this document by reference with respect to suitable vectors for the expression of a gene of interest. Certain vectors that can be used for the expression of transgenes as described in this document include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for the expression of a transgene contain polynucleotide sequences that enhance the translation rate of these genes or improve the nuclear stability or export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, an internal ribosome entry site (IRES), and a polyadenylation signal site that directs efficient transcription of the gene carried in the expression vector.The expression vectors suitable for use with the compositions and methods described in this document may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of a suitable marker are genes encoding antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, nourseotricin, or zeocin.

[0155] As used herein, the term “isolate” refers to a substance or entity that is altered or removed from its natural state (e.g., altered or removed from at least some component with which it is associated in its natural state). For example, a nucleic acid or peptide naturally present in a living animal is not isolated, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is isolated. An isolated protein or nucleic acid may exist in a substantially purified form or may exist in a non-native environment, such as, for example, in a transgenic host cell. Such polynucleotides may be part of a vector and / or such polynucleotides or polypeptides may be part of a composition and still be isolated, since such vector or composition is not part of it. Petition 870250096842, dated 10 / 23 / 2025, p. 51 / 180 44 / 121 of the environment in which it is found in nature. In some embodiments, an isolated nucleic acid is recombinant (e.g., incorporated into a vector). Isolation methods for polynucleotides and proteins or polypeptides are routine in the technique.

[0156] As used herein, the terms “natural occurrence” or “wild type” mean existing in nature without artificial assistance or human involvement. “Natural occurrence” or “wild type” may refer to a native form of a biomolecule, sequence, or entity.

[0157] As used herein, the phrase operably linked refers to a functional link between two or more similar molecules, constructs, transcripts, entities, or portions. For example, a DNA segment may be operatively linked to another DNA segment if they are positioned relative to each other on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding region so as to facilitate transcription of the coding region. In other examples, operatively linked nucleic acids are not contiguous but are positioned in such a way that they have a functional relationship to each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, need not be contiguous. Linkage may be achieved by ligation at convenient restriction sites or by the use of synthetic oligonucleotide adapters or ligands.

[0158] As used herein, the term “contact” (i.e., bringing a cell into contact with an agent) is intended to include incubating the agent and the cell together in vitro (e.g., adding the agent to cells in culture) or administering the agent to a subject so that the agent and the subject’s cells are brought into contact in vivo. The term “contact” is not intended to include exposure of cells to an agent that may occur naturally in Petition 870250096842, dated 10 / 23 / 2025, page 52 / 180 45 / 121 a subject (i.e., exposure that may occur as a result of a natural physiological process).

[0159] As used herein, the terms associated with, conjugated, linked, attached, and fastened, when used with respect to two or more portions, mean that the portions are physically associated or linked to each other, either directly or through one or more additional portions that serve as a linking agent, to form a structure that is sufficiently stable that the portions remain physically associated under the conditions in which the structure is used, for example, physiological conditions. An association need not be strictly by means of direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a connectivity based on hybridization sufficiently stable such that the associated entities remain physically associated.

[0160] As used herein, the term “stability” in reference to biological material or a molecule (e.g., a polynucleotide or a polypeptide) refers to the balance between production (e.g., transcription or translation) and decay or degradation or the steady-state levels of the biological material in a system, such as a whole organism, an organ, a tissue or subset of tissues, a cell or subset of cells, or in a plate or receptacle. In some embodiments, stability refers to the half-life of the biological material or molecule.

[0161] As used in this document, the term cell type refers to a group of cells that share a phenotype that is statistically separable based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include those isolated from a common tissue. Petition 870250096842, dated 10 / 23 / 2025, page 53 / 180 46 / 121 (for example, epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those that are isolated from a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0162] As used in this document, the term “in vitro” refers to events that occur in an artificial environment, for example, in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than inside an organism (e.g., animal, plant, or microbe).

[0163] As used in this document, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe, or a cell or tissue thereof).

[0164] As used herein, the term “ex vivo” refers to events that occur to a component of an organism (e.g., a tissue, a cell, or a subcellular fraction) when it is removed from its natural environment (e.g., the body or natural structure) and placed in an artificial environment (e.g., a test tube or culture plate, flask, or other receptacle) for experimental or clinical applications. In some cases, ex vivo experimentation or applications may involve the administration (e.g., implantation, injection, depot, infusion, among other suitable routes of administration) of the component to the same subject or to a separate recipient subject after one or more ex vivo applications.

[0165] As used herein, “modified” refers to an altered state or structure of a molecule (e.g., a polynucleotide; e.g., DNA or mRNA; e.g., a polypeptide or protein; e.g., an amino acid residue) of the invention. Molecules can be modified in many ways, such as structural modifications (e.g., mutation of one or more base pairs or amino acid residues) or chemical modifications (e.g., methylation, acetylation, reduction or Petition 870250096842, dated 10 / 23 / 2025, page 54 / 180 47 / 121 oxidation, glycosylation, lipidation, ubiquitination of one or more base pairs or amino acid residues). In some embodiments, a molecule such as DNA or mRNA is modified to remove, reduce, or eliminate DRACH motifs to reduce the number of m6A methylation modifications in a gene or coding sequence of interest.

[0166] As described in this document, the term exogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found in nature in a particular organism (e.g., a human being) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are supplied from a source external to an organism or to cultured matter extracted from it.

[0167] As used in this document, the term homology refers to the overall relationship between polymeric molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered homologous to each other if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical.In some embodiments, polymeric molecules are considered homologous to each other if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar. The term homologous refers to... Petition 870250096842, dated 10 / 23 / 2025, page 55 / 180 48 / 121 necessarily involves a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0168] Percentage (%) of sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining the percentage of nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of a person skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2 or Megalign software.Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment with respect to the total length of the sequences being compared. For example, percentage sequence identity values ​​can be generated using the BLAST sequence comparison computer program. By way of illustration, the percentage sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which may alternatively be expressed as a given nucleic acid or amino acid sequence, A, having a certain percentage sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by an alignment program. Petition 870250096842, dated 10 / 23 / 2025, p. 56 / 180 49 / 121 sequence (e.g., BLAST) in the alignment of this program A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percentage of sequence identity from A to B will not be equal to the percentage of sequence identity from B to A.

[0169] In certain embodiments, the term “reference level” herein refers to a value from a “reference sample” for determining the effect induced by the methods described herein. A reference level may be a metric or measurement determined prior to administration or implementation of the method (e.g., one or more codon optimization methods or one or more treatment methods described herein). A reference level may be a metric or measure determined in a reference sample in which the methods described herein have not been administered (e.g., a negative control sample; e.g., a healthy subject control or a subject with a disease or condition).A reference level can be a metric or measure determined on a reference sample that exhibits a known or expected effect for evaluating the effectiveness of the methods described herein (e.g., effects produced by routine protein production methods or effects produced by known treatment methods). In some embodiments, a reference level may be a predetermined value or a value. As the skilled practitioner will be able to perceive, the reference level is predetermined and defined to meet requirements in terms of, for example, specificity and / or sensitivity. It may be, for example, that the sensitivity or specificity of the assay, respectively, have to be defined within certain limits, e.g., 80%, 90%, or 95%. These requirements may also be defined in terms of positive or negative predictive values. In one embodiment, the reference level is determined at... Petition 870250096842, dated 10 / 23 / 2025, page 57 / 180 50 / 121 healthy individuals. The reference value in a modality was predetermined in the disease entity to which the subject belongs. In certain modalities, the reference level may be defined as any percentage between, for example, 25% and 75% of the overall distribution of values ​​in an investigated disease entity. In other modalities, the reference level may be defined as, for example, the median, tertiles, quartiles, or quintiles, as determined from the overall distribution of values ​​in an investigated disease entity or in a given population. In one modality, the reference level is defined as the median value determined from the overall distribution of values ​​in an investigated disease entity. In some modalities, the reference level may depend on the patient's sex, for example, men may have a different reference level than women.

[0170] As used herein, the term “sample” refers to a subset of its tissues, cells, or component parts (e.g., body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretions, mucous secretion, fecal water, pancreatic juice, sinus cavity lavage fluids, bronchopulmonary aspirates, blastocillic cavity fluid, and umbilical cord blood).A sample may also include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including, but not limited to, for example, plasma, serum, fluid. Petition 870250096842, dated 10 / 23 / 2025, page 58 / 180 51 / 121 spinal, lymphatic fluid, the outer sections of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. An example also refers to a medium, such as a broth or nutrient gel, that may contain cellular components, such as proteins or nucleic acid molecules.

[0171] As used in this document, treatment and treating, in reference to a disease or condition, refer to an approach to achieving beneficial or desired outcomes, for example, clinical outcomes. Beneficial or desired outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions; reduction in the extent of the disease or condition; stabilization of the disease, disorder, or condition (i.e., without worsening); prevention of the spread of diseases or conditions; delaying or slowing the progression of the disease or condition; improvement or palliation of the disease or condition; and detectable or undetectable remission (partial or total). “Improvement” or “alleviation” of a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are reduced and / or the course of progression is slowed or prolonged, compared to the extent or time course in the absence of treatment."Treatment" can also mean prolonging survival compared to the expected survival without treatment. Those who need treatment include those who already have the condition or disorder, as well as those likely to have the condition or disorder, or those in whom the condition or disorder should be prevented.

[0172] As used herein, the terms “effective amount”, “therapeutically effective amount” and “sufficient amount” of a composition described herein refer to an amount sufficient to, when administered to the subject (e.g., a mammal; e.g., a human subject), produce beneficial or desired results, including results Petition 870250096842, dated 10 / 23 / 2025, page 59 / 180 52 / 121 clinical cases and, as such, an “effective amount” or synonym thereof depends on the context in which it is being applied. The amount of a given composition described herein that will correspond to such an amount will vary depending on a number of factors, such as the therapeutic agent in question (e.g., polynucleotide, transgene or coding sequence), the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or the host to be treated, and the like, but may nevertheless be routinely determined by a specialist in the field. Furthermore, as used herein, a therapeutically effective amount of a composition of the present disclosure is an amount that results in a beneficial or desired outcome in a subject compared to a reference (e.g., the subject before treatment, a healthy control or an untreated subject).As defined herein, a therapeutically effective amount of a composition of the present disclosure can be readily determined by a practitioner with ordinary knowledge using routine methods known in the field. A dosage regimen can be adjusted to provide the optimal therapeutic response.

[0173] As used herein, “administration” refers to the distribution, delivery, or application of a disclosure composition to a subject by any route suitable for delivery of the composition (e.g., a polynucleotide; for example, a polynucleotide encoding a transgene), to the desired location in the subject. Exemplary routes of administration include intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intra-arterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous administration.

[0174] As used herein, the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within such an interval. Petition 870250096842, dated 10 / 23 / 2025, p. 60 / 180 53 / 121 that there may be an overlap of the effect of each agent on the subject who needs it. In some modalities, the agents are administered at intervals of about 1 or more weeks, 1 or more days, 1 or more hours, or 1 or more minutes apart. In some modalities, the administrations of the agents are spaced sufficiently close to each other so that a combination (e.g., a synergistic effect) is achieved.

[0175] As used in this document, the term “pharmaceutically acceptable” refers to compounds, materials, compositions and / or dosage forms that are, within the scope of good medical sense, suitable for use in contact with the tissues of a subject, such as a mammal (e.g., a human), without excessive toxicity, irritation, allergic response or other problem or complication, consistent with a reasonable benefit / risk ratio.

[0176] As used herein, the term “pharmaceutically acceptable excipient” refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions that has the properties of being substantially non-toxic and non-inflammatory in individuals.

[0177] As used in this document, the term pharmaceutical composition refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers, to be administered to a subject, such as a mammal, for example, a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject. DETAILED DESCRIPTION

[0178] Compositions and methods for use in codon optimization technology are described here. For example, the compositions and methods described here are aimed at improving the stability and half-life of Petition 870250096842, dated 10 / 23 / 2025, page 61 / 180 54 / 121 mRNA for improved production of exogenous proteins.

[0179] Advantageously, the methods described herein allow for improved production of recombinant or exogenous proteins by modifying polynucleotide sequences that encode the protein of interest, such that m6A methylation of the transcribed mRNA sequence is reduced, thereby increasing mRNA stability and producing more robust protein expression compared to traditional protein production methods. For example, the methods described herein can be applied to express proteins in vitro, in vivo, and / or ex vivo for therapeutic purposes, such as gene therapy, drug delivery, and / or vaccine development. I. Codon optimization methods

[0180] The codon optimization methods described here can be used alone or in combination to improve protein expression in applications targeting transgene expression or heterologous gene expression. In general, codon optimization improves the efficiency of translation of a target gene or transcript into a polypeptide or protein product for higher protein yield. The codon optimization methods described here can be applied to a gene or polynucleotide sequence encoding any type of protein, including a soluble protein, a transmembrane protein, a membrane-associated protein, an intracellular protein, or a secreted protein, among others.In some embodiments, the codon optimization methods described herein are applied to a gene or polynucleotide sequence that encodes an entire protein (e.g., the complete amino acid sequence), a polypeptide, or a protein fragment (e.g., one or more protein domains, a polypeptide of a protein, and / or one or more protein chains). In other embodiments, the codon optimization methods described herein are applied to a gene or polynucleotide sequence. Petition 870250096842, dated 10 / 23 / 2025, page 62 / 180 55 / 121 which encodes a fusion protein (e.g., an Fc fusion protein or an albumin fusion protein), which can further increase the stability of the protein and / or the half-life of the protein. A. Reduction or elimination of motives DRACH

[0181] In the methods described herein, increased protein expression of a protein of interest can be achieved by codon optimization techniques that reduce or eliminate DRACH motifs in a gene or coding sequence of a target of interest. A DRACH motif refers to a short five-base-pair nucleic acid consensus sequence, where “D” designates guanine, adenine, or uracil; “R” designates guanine or adenine; “A” designates adenine; “C” designates cytosine; and “H” designates adenine, uracil, or cytosine. DRACH motifs include the following unique sequences: AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, TGACT, AAACA, AAACC, AAACT, AGACC, GAACA, GGACC, TAACA, TAACC, TAACT, TGACA, and TGACC.

[0182] A DRACH motif is more likely to undergo N(6)-methyladenosine (“m6A”) modification at the adenosine base at the third position, which in turn reduces the half-life of a target mRNA in a cell or biological sample containing mRNA. By modifying a gene or coding sequence to reduce or eliminate one or more DRACH motifs, the resulting mRNA transcript may have an increased half-life due to fewer m6A modifications compared to the unmodified gene or coding sequence. In some embodiments, the increased half-life of modified mRNA transcripts may be due to increased stability or reduced degradation. For example, proteins of the YTHDF protein family (e.g., YTHDF2 and YTHDC2) recognize m6A-modified mRNA transcripts in the cytosol and mediate the degradation of these modified transcripts by destabilizing the m6A-modified mRNA transcripts and / or recruiting Petition 870250096842, dated 10 / 23 / 2025, page 63 / 180 56 / 121 exonucleases.

[0183] A DRACH motif has a higher propensity or probability of receiving an m6A modification, as demonstrated by methylation enrichment studies. The propensity of a DRACH motif to undergo an m6A modification may depend on the primary sequence of the motif, the location of the motif in the gene or coding sequence, the topology of the gene or sequence, such as secondary or tertiary structural elements around the motif, or a combination thereof. For example, one or more of these factors may increase the accessibility of the DRACH sequence to a methyltransferase, such as METTL3, METTL14, WTAP, KIAA1439, METTL16, RBM15, and / or ZC3H13 for m6A modification. A DRACH motif with the nucleotide sequence selected from the AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, and TGACT groups has a higher probability of undergoing an m6A modification. Sequence-dependent enrichment of m6A modifications in DRACH motifs has been discussed elsewhere, such as in Schwartz et al. (Cell Rep.8(1): 284-296, 2014), incorporated here by reference. Polynucleotide topology and sequence elements also influence the enrichment of m6A-modified DRACH motifs in an mRNA transcript. For example, m6A modifications can be enriched in DRACH motifs in the coding sequence of a gene, in the 3' untranslated region and / or near stop codons. In contrast, a DRACH motif has a lower propensity to undergo m6A modification if the DRACH motif is closer to a splice junction or in the 5' untranslated region. In some embodiments, a DRACH motif is less likely to receive an m6A modification if it is about 50 nucleotides or less (e.g., about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides) from a splice junction. Petition 870250096842, dated 10 / 23 / 2025, p. 64 / 180 57 / 121

[0184] The presence of an m6A modification in a DRACH motif of an RNA molecule can be detected based on methods known in the art, such as RNA crosslinking and immunoprecipitation techniques, coupled with next-generation sequencing modalities, as well as nanopore sequencing. Methods for detecting m6A modifications in mRNA transcripts are known in the art and have been described in detail in, for example, Meyer et al. (Cell. 149(7): 1635-1646, 2012). Dominissini et al. (Nature. 29(485): 201-206, 2012), Linder et al. (Nat Methods. 12(8): 767-772, 2015), Ke et al. (Genes Dev. 31(10):990-1006, 2017) and Lorenz et al. (RNA. 26: 19-28, 2020), each of which is incorporated herein by reference.

[0185] In some embodiments, a gene or coding sequence is modified so that between 1 and 5 DRACH motifs (e.g., 1, 2, 3, 4, or 5 DRACH motifs) are removed from a gene or coding sequence. In some embodiments, a gene or coding sequence is modified so that between 5 and 10 DRACH motifs (e.g., 5, 6, 7, 8, 9, or 10 DRACH motifs) are removed from a gene or coding sequence. In some embodiments, a gene or coding sequence is modified so that between 10 and 20 DRACH motifs (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 DRACH motifs) are removed from a gene or coding sequence.In some embodiments, a gene or coding sequence is modified so that between 20 and 40 DRACH motifs (e.g., 20 to 25, 25 to 30, 30 to 35, 30 to 40; e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 DRACH motifs) are removed from a gene or coding sequence. In some embodiments, a gene or coding sequence is modified so that between 40 and 80 DRACH motifs (e.g., 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80; e.g., 41, 42, 43, 44, 45,...) Petition 870250096842, dated 10 / 23 / 2025, p. 65 / 180 58 / 121 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 or 80 DRACH motifs are removed from a gene or coding sequence. In some embodiments, a gene or coding sequence is modified so that between 50 and 100 DRACH motifs (e.g., 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100; e.g., 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, or 100 DRACH motifs are removed from a gene or coding sequence. In other embodiments, more than 100 DRACH motifs (e.g., more than 100, more than 110, more than 120, more than 130, more than 140, more than 150, more than 160, more than 170, more than 180, more than 190, or more than 200 DRACH motifs) are removed from a gene or coding sequence.

[0186] A gene or coding sequence may be modified so that all DRACH motifs are removed or eliminated from a gene or coding sequence. In some embodiments, a gene or coding sequence is modified so that the number of DRACH motifs in a sequence is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, or at least 10% compared to the native gene or coding sequence.

[0187] Methods for removing one or more DRACH motifs can be combined with additional codon usage practices to further reduce the likelihood of m6A modification of a sequence. In some embodiments, the additional codon usage practice to reduce m6A modification employs one or more of the following six guidelines: (1) never use GAC for aspartic acid (Asp or D); (2) never use GAA for glutamic acid (Glu or E); (3) never use GGA for glycine (Gly or G); (4) never use AAA for lysine (Lys). Petition 870250096842, dated 10 / 23 / 2025, p. 66 / 180 59 / 121 or K); (5) never use AAC for asparagine (Asn or N); and / or (6) always use ACG for threonine (Thr or T).

[0188] In some embodiments, the removal of one or more DRACH motifs requires iterative modification of a polynucleotide of interest to avoid the introduction of new DRACH motifs and / or to preserve the identity of the amino acid sequence. In some embodiments, the removal of one or more DRACH motifs involves the substitution of one or more codons in the polynucleotide sequence with a synonymous codon (i.e., a codon that codes for the same amino acid sequence). In some embodiments, the removal of one or more DRACH motifs involves the substitution of nucleotides so that the primary sequence (i.e., amino acid sequence) of a polypeptide or protein has 100% sequence identity.

[0189] Other considerations that may influence mRNA stability or total protein production include mRNA GC content, mRNA secondary structure, accessibility to ribosomal binding sites, relative tRNA abundance, among other considerations known in the art. In some embodiments, a higher GC content in a coding sequence improves mRNA stability and half-life by inhibiting mRNA decay and repression mechanisms in the cell (e.g., localization in P-body granules or reduced interactions with translational repressors). The ideal GC content for improved protein production may be between 50% and 80% GC content (e.g., between 50-60%, between 60-70%, or between 70-80%; for example, 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%, or 80%) along the length of the coding sequence of interest.Structural considerations for an mRNA transcript include secondary structures, such as hairpins or stem loops, which can inhibit the initiation of translation. Petition 870250096842, dated 10 / 23 / 2025, page 67 / 180 60 / 121 or promote ribosome arrest. Clamp or stem loops can form when intramolecular hydrogen bonds form between complementary nucleotides (e.g., A and T, A and U, or G and C). The mechanisms of ribosome scanning and ribosome fidelity that influence protein production are known in the art and are discussed elsewhere, such as in Zaher and Green (Cell. 136(4): 746-762, 2009), which is incorporated here by reference. The codon optimization method in which one or more DRACH motifs are removed can be performed iteratively to achieve an optimal value of any or a combination of the above considerations. In some embodiments, a codon optimization method is performed 1 time, 2 times, 3 times, or more than 3 times to achieve the ideal mRNA GC content and structural considerations.

[0190] In some embodiments, codon optimization by removing or eliminating one, more than one, or all of the DRACH motifs present leads to an increase in the mRNA half-life (e.g., improved stability) of a gene or coding sequence of interest. In some embodiments, the elimination of one, more than one, or all of the DRACH motifs present in a gene or coding sequence of interest increases the mRNA half-life by approximately 1.25 times, approximately 1.5 times, approximately 1.75 times, approximately 2 times, approximately 2.25 times, approximately 2.5 times, approximately 2.75 times, approximately 3 times or more, compared to a polynucleotide sequence in which the DRACH motifs have not been removed, such as the native gene or coding sequence.In some embodiments, the elimination of one, more than one, or all DRACH motifs present in a gene or coding sequence of interest increases the mRNA half-life by about 5 times to about 50 times (e.g., by about 5 times, by about 6 times, by about 7 times, by about 8 times, by about 9 times, by about 10 times, by about 15 times, by about 5 times). Petition 870250096842, dated 10 / 23 / 2025, p. 68 / 180 61 / 121 of 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times or more than 50 times), compared to a polynucleotide sequence in which the DRACH motifs have not been removed, such as the native gene or coding sequence.

[0191] In some embodiments, the elimination of one, more than one, or all of the DRACH motifs present in a gene or coding sequence of interest increases the mRNA half-life by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100%, compared to a polynucleotide sequence in which the DRACH motifs have not been removed, such as the native gene or coding sequence.In some embodiments, the elimination of one, more than one, or all DRACH motifs present in a gene or coding sequence of interest increases the mRNA half-life, for example, by about 1 to 24 hours, or more (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 24 hours or more), compared to a polynucleotide sequence in which the DRACH motifs have not been removed, such as the native gene or coding sequence. Methods for measuring mRNA half-life or stability include those known in the art and described herein.

[0192] In some embodiments, the elimination of one, more than one, or all DRACH motifs present in a gene or coding sequence of interest increases the production of a protein or fragment thereof (e.g., protein expression). In some embodiments, the elimination of one, more than one, or all DRACH motifs present in a gene or coding sequence of interest increases protein expression by approximately 1.5 times, Petition 870250096842, dated 10 / 23 / 2025, page 69 / 180 62 / 121 in about 2 times, in about 3 times, in about 4 times, in about 5 times or more than 5 times, compared to a polynucleotide sequence in which the DRACH motifs have not been removed, such as the native gene or coding sequence of interest.In some embodiments, the elimination of one, more than one, or all DRACH motifs present in a gene or coding sequence of interest increases protein expression by about 5-fold to about 50-fold (e.g., about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or more than 50-fold), compared to a polynucleotide sequence in which the DRACH motifs have not been removed, such as the native gene or coding sequence.

[0193] In some embodiments, the elimination of one, more than one, or all DRACH motifs present in a gene or coding sequence of interest increases protein expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100%, compared to a polynucleotide sequence in which the DRACH motifs have not been removed, such as the native gene or coding sequence. In some embodiments, an increase in the expression of a protein of interest or a fragment thereof is defined by an increase in the total protein yield from recombinant production or an increase in the protein measured in a biological sample. Methods for measuring protein expression are known in the art and are described herein. B. Codon optimization based on codon usage bias

[0194] In some modalities, a gene expression method Petition 870250096842, dated 10 / 23 / 2025, page 70 / 180 63 / 121 heterologous gene expression by reduction or elimination of DRACH motifs may also include codon optimization based on codon usage bias. Codon usage bias refers to the biological phenomenon in which there is preferential use of a specific codon over other synonymous codons, which can influence the rates and efficiency of protein translation. Codon usage bias can influence RNA processing, ribosomal scanning, translation initiation or termination, and / or protein folding and stability. Codon usage bias varies depending on the organism encoding the sequence due to evolutionary impacts on an organism's genome, including mutations, recombination rates, and genetic drift. Variations in codon usage are observed in organisms of different species, families, or groups, and even in different genes within the same species. Therefore, codon usage bias can be considered for heterologous gene expression applications.For example, a polynucleotide sequence native to a human cell may produce less abundant protein when produced in an Escherichia coli cell compared to a human cell if it is not first modified based on the use of codons in E. coli.

[0195] In some embodiments, codon optimization for heterologous gene expression involves the use of a codon usage frequency table for a given organism. An example of a codon usage frequency table for a human is shown in Table 1 below. Table 1: Frequency table of exemplary codon usage Codon Amino Acid Frequency Codon Amino Acid Frequency TGA TAA * * 0.52 0.28 ATG M (Met) 1.0 TAG * 0.20 AAC AAT N (Asn) N (Asn) 0.54 0.46 GCC A (Ala) 0.40 CCC P (Pro) 0.33 GCT A (Ala) 0.26 CCT P (Pro) 0.28 GCA A (Ala) 0.23 CCA P (Pro) 0.27 GCG A (Ala) 0.11 CCG P (Pro) 0.11 Petition 870250096842, dated 10 / 23 / 2025, page 71 / 180 64 / 121 Códon Aminoácido Frequência Códon Aminoácido Frequência TGC C (Cys) 0.55 CAG Q (Gln) 0.75 TGT C (Cys) 0.45 CAA Q (Gln) 0.25 GAC D (Asp) 0.54 CGG R (Arg) 0.21 GAT D (Asp) 0.46 AGA R (Arg) 0.20 GAG E (Glu) 0.58 AGG R (Arg) 0.20 GAA E (Glu) 0.42 CGC CGA R (Arg) R (Arg) 0.19 0.11 TTC TTT F (Phe) F (Phe) 0.55 0.45 CGT R (Arg) 0.08 AGC S (Ser) 0.24 GGC G (Gly) 0.34 TCC S (Ser) 0.22 GGA G (Gly) 0.25 TCT S (Ser) 0.18 GGG G (Gly) 0.25 AGT S (Ser) 0.15 GGT G (Gly) 0.16 TCA S (Ser) 0.15 CAC H (His) 0.59 TCG S (Ser) 0.06 CAT H (His) 0.41 ACC T (Thr) 0.36 ATC I (Ile) 0.48 ACA T (Thr) 0.28 ATT I (Ile) 0.36 ACT T (Thr) 0.24 ATA I (Ile) 0.16 ACG T (Thr) 0.12 AAG K (Lis) 0.58 GTG V (Val) 0.47 AAA K (Lis) 0.42 GTC V (Val) 0.24 CTG L (Leu) 0.41 GTT V (Val) 0.18 CTC L (Leu) 0.20 GTA V (Val) 0.11 TTG L (Leu) 0.13 TGG W (Tryp) 1.0 CTT L (Leu) L (Leu) 0.13 0.07 TTA TAC Y (Tyr) 0.57 CTA L (Leu) 0.07 TAT Y (Tyr) 0.43 Códon: A = adenine, C = cytosine, G = guanine, T = thymine; Amino acid: * = stop codon, A = alanine, C = cysteine, D = aspartic acid, E = glutamic acid; F = phenylalanine; G = glycine, H = histidine, I = isoleucine, K = lysine, L = leucine, M = methionine, N = asparagine, P = proline, Q = glutamine, R = arginine, S = serine, T = threonine, V = valine, W = tryptophan, Y = tyrosine; The reported frequency corresponds to human codons.

[0196] A method of codon optimization to increase protein expression of a target of interest can be achieved by modifying the gene or coding sequence to replace one or more codons with the synonymous codon with the highest frequency of use. For example, a gene or coding sequence for protein expression in a human can be modified so that only the codons with the highest frequency, as shown in Table 1, are represented in each instance for a specific amino acid in the modified sequence (e.g., all the Petition 870250096842, dated 10 / 23 / 2025, page 72 / 180 65 / 121 codons that code for leucine are modified to CTG.

[0197] In some embodiments, a gene or protein expression coding sequence can be modified so that all recited codons are replaced by synonymous codons that have the highest frequency of use in an organism. In some embodiments, a gene or protein expression coding sequence can be modified so that between 5% and 50% of the recited codons (e.g., between 5% and 10%, between 10% and 25%, between 20% and 40%, or between 25% and 50%; for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%) are replaced by synonymous codons that have the highest frequency of use.In some embodiments, a gene or protein expression coding sequence can be modified so that between 25% and 75% (for example, between 25% and 40%, between 30% and 50%, between 40% and 60%, or between 50% and 75%; for example, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%) of the recited codons are replaced by synonymous codons that have the highest frequency of use. In some embodiments, a gene or protein expression coding sequence can be modified so that between 75% and 100% (for example, between 75% and 85%, between 80% and 90%, between 85% and 95%, between 90% and 100%; for example, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) of the recited codons are replaced by synonymous codons that have the highest frequency of use.

[0198] A method of codon optimization to increase the protein expression of a target of interest can be achieved by modifying the gene or codon sequence so that one or more codons are represented proportionally to their reported frequency of use in a Petition 870250096842, dated 10 / 23 / 2025, p. 73 / 180 66 / 121 organ. For example, a sequence of genes or codons for protein expression in a human being can be modified so that the codons encoding a specific amino acid are represented according to their frequency of use, as shown in Table 1 (for example, the codons encoding leucine are modified so that CTG is represented in about 41% of cases, CTC is represented in about 20% of cases, TTG is represented in about 13% of cases, etc.).

[0199] In some embodiments, a gene or protein expression coding sequence can be modified so that all recited codons are replaced by synonymous codons based on their proportional frequency of use. In some embodiments, a gene or protein expression coding sequence can be modified so that between 5% and 50% of the recited codons (e.g., between 5% and 10%, between 10% and 25%, between 20% and 40%, or between 25% and 50%; for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%) are replaced by synonymous codons based on their proportional frequency of use.In some embodiments, a gene or protein expression coding sequence can be modified so that between 25% and 75% (e.g., between 25% and 40%, between 30% and 50%, between 40% and 60%, or between 50% and 75%; for example, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%) of the recited codons are replaced by synonymous codons based on their proportional frequency of use. In some embodiments, a gene or coding sequence for protein expression can be modified so that between 75% and 100% (e.g., between 75% and 85%, between 80% and 90%, between 85% and 95%, between 90% and 100%; e.g., about 75%, about 80%, about 85%, about 90%, about 90%, about 90%, about 95 ... Petition 870250096842, dated 10 / 23 / 2025, p. 74 / 180 67 / 121 95% or approximately 100% of the recited codons should be replaced by synonymous codons based on their proportional frequency of use.

[0200] In some embodiments, the codon optimization method is performed using available software or web tools known in the art, where codon optimization is automated or performed based on an algorithm. In some embodiments, the codon optimization method involves a heuristic scoring method. In some embodiments, the codon optimization method involves the use of a neural network. In some embodiments, the codon optimization method is performed with quantum computing (see, for example, Fox et al. PLoS ONE 16(10):e0259101, 2021, incorporated herein by reference). In some embodiments, a codon optimization method is performed iteratively to achieve the ideal guanine-cytosine (GC) content of the mRNA, mRNA secondary structure, mRNA motifs, ribosomal binding sites, among other considerations. C. Methods for evaluating codon optimization

[0201] A codon optimization method can be evaluated by measuring the mRNA stability or half-life of the modified gene or coding sequence compared to the native gene or coding sequence. In some embodiments, one or more codon optimization methods described herein, or a combination thereof, produce an mRNA transcript with greater stability or a longer half-life. In some embodiments, the mRNA half-life is evaluated by one or more measurements of the concentration, relative abundance, or relative stability of an mRNA transcript detected in a biological sample. The concentration, relative abundance, or relative stability of an mRNA transcript can be evaluated by reverse transcription polymerase chain reaction (RT-PCR), fluorimetry, colorimetry, spectrophotometric analyses, flow cytometry, hybridization methods in Petition 870250096842, dated 10 / 23 / 2025, page 75 / 180 68 / 121 situ, Northern blot analyses, or a matrix using one or more hybridizing oligonucleotides or antibodies against one or more mRNA transcripts of interest. In other embodiments, the half-life or stability of an mRNA transcript of interest is evaluated by structural or biochemical methods. In some embodiments, the half-life or stability of an mRNA transcript is evaluated by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) or modified methodologies (see, for example, Wilkinson et al. (Nat. Prot. 1: 1610-1616, 2006, incorporated herein by reference). In other embodiments, the half-life or stability of an mRNA transcript of interest may be evaluated by mass spectrometry to assess the presence of chemical modifications. One or more methods of evaluating mRNA half-life or stability, as described herein, may be used in any combination, optionally in addition to other methods known in the art.

[0202] In some embodiments, the half-life or stability of an mRNA transcript of interest is assessed by pulse chasing methodologies to analyze mRNA degradation. In some embodiments, the mRNA half-life of an mRNA transcript of interest is assessed by measuring the mRNA decay rate after transcriptional inhibition (e.g., by applying actinomycin D to a biological sample where the biological sample is derived from a subject or from a cultured cell population in vitro). In any of the foregoing embodiments, the stability or half-life of an mRNA transcript of a modified gene or coding sequence can be compared to a reference sample. In some embodiments, the reference sample is derived from the native gene or coding sequence.In other modalities, the reference sample is a maintenance gene (e.g., a gene associated with viability that is stably expressed in a cell; e.g., beta-actin, GAPDH, rRNA). Petition 870250096842, dated 10 / 23 / 2025, p. 76 / 180 69 / 121

[0203] Additionally or alternatively, the codon optimization method can be evaluated by measuring the protein production by the modified gene or coding sequence, compared to the native gene or coding sequence. In some embodiments, protein production is evaluated by measuring the concentration of a protein of interest in a sample. In some embodiments, the concentration of a protein of interest is measured by mass spectrometry, Western blot analysis, ELISA, immunoprecipitation methods, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or an array using binding oligonucleotides, such as aptamers or antibodies against one or more proteins of interest.

[0204] The codon optimization method can be evaluated based on the quality of the protein or the stability of the produced protein or polypeptide of interest. Protein quality or stability can be measured by functional or binding assays specific to the protein of interest (e.g., an enzymatic activity assay or a binding assay using one or more known binding partners as a ligand or analyte). Protein quality or stability can also be evaluated using biochemical or biophysical methods known in the art, such as, for example, differential scanning fluorimetry, thermal shift assays, circular dichroism, dynamic light scattering, analytical ultracentrifugation, size exclusion chromatography, isothermal titration calorimetry, microscale thermophoresis, mass spectrometry, hydrogen-deuterium exchange, among other methods known in the art (see, for example, Le Basle et al. (J. Pharm.Sci. 109(1): 169-190, 2020), incorporated here by reference). II. Applications of methods for codon optimization A. Protein production method

[0205] Any of the codon optimization methods Petition 870250096842, dated 10 / 23 / 2025, p. 77 / 180 The 70 / 121 embodiments described herein can be used alone or in combination for heterologous or transgenic gene expression in a host cell, such as a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. In other embodiments, the host cell is cultured in vitro for heterologous gene expression or recombinant protein production. In some embodiments, the host cell is cultured as an adherent cell or as a cell in suspension. In some embodiments, the host cell is an established model cell line, such as a cell line for culture available through the American Type Culture Collection (ATCC).

[0206] In some embodiments, the host cell is in an organism for the production of a protein in vivo (e.g., transgenic expression). In some embodiments, the host cell is derived from an organism (e.g., a human subject) for in vitro culture methods, whereby after in vitro culture, the host cell can be implanted, injected, deposited, or otherwise administered to the same organism or a different organism (e.g., for autologous or allogeneic therapies or treatments). B. Codon-optimized gene product delivery methods

[0207] A codon-optimized gene product (e.g., a transgene, an mRNA transcript, or a purified or partially purified protein) can be delivered to a host cell or subject via a variety of delivery techniques. The following sections describe exemplary and non-limiting modalities for delivering a polynucleotide of interest (e.g., a polynucleotide in which one or more DRACH motifs have been eliminated according to the methods described herein) to a host cell or subject. Petition 870250096842, dated 10 / 23 / 2025, page 78 / 180 71 / 121 i. Viral genomes for codon-optimized gene product delivery

[0208] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a gene of interest into the genome of a target cell (e.g., a mammalian cell, such as a human cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require added proteins or reagents to induce gene integration.Examples of viral vectors that can be used in conjunction with the compositions and methods described herein are adeno-associated viruses (AAVs), retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified Ankara vaccinia (MVA), avian pox, and canary pox). Other viruses that can be used in conjunction with the compositions and methods described herein include Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalian type C viruses, type B viruses, type D viruses, HTLV-BLV group, lentiviruses, foam viruses (Coffin, JM, Retroviridae: The Viruses and Their Replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include viruses of... Petition 870250096842, dated 10 / 23 / 2025, p. 79 / 180 72 / 121 murine leukemia virus, murine sarcoma virus, mouse mammalian tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, endogenous baboon viruses, gibbon primate leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, disclosure of which is incorporated herein by reference with respect to viral vectors for use in gene therapy. ii. AAV vectors for codon-optimized gene product delivery

[0209] In some embodiments, a codon-optimized polynucleotide (e.g., a modified gene or coding sequence) described herein is incorporated into the recombinant AAV (rAAV) vector to facilitate introduction into a cell. Useful rAAV vectors in conjunction with the compositions and methods described herein include recombinant nucleic acid constructs containing (1) a transgene encoding a codon-optimized sequence for enhanced protein production and (2) one or more nucleic acids that facilitate expression of the modified gene or coding sequence. The viral nucleic acids may include those cis-acting elements of the rAAV for replication and packaging (e.g., functional inverted terminal repeats, or “ITRs”) of DNA into a virion. Such rAAV vectors may also contain marker or reporter genes.Useful rAAV vectors include those that have one or more naturally occurring AAV genes deleted in whole or in part, but retain functional flanking ITR sequences. AAV ITRs can be of any serotype (e.g., derived from serotype 2) suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al. (J. Biomed. Sci. 7:279-291, 2000), and Monahan and Samulski (Gene Delivery. 7:24-30, 2000), the publications of each. Petition 870250096842, dated 10 / 23 / 2025, page 80 / 180 73 / 121 one of them are incorporated here by reference, as they belong to AAV vectors for gene delivery.

[0210] The nucleic acids and vectors described in this document can be incorporated into an rAAV virion in order to facilitate the introduction of the nucleic acid or vector into a cell. The AAV capsid proteins comprise the non-nucleic acid outer portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, which are necessary for virion assembly. The construction of rAAV virions has been described, for example, in US Patents Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), each of whose disclosures is incorporated herein by reference with regard to AAV vectors for gene delivery.

[0211] Useful rAAV virions in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, and 9, among others. The construction and use of AAV vectors and AAV proteins from different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623, 2000), Davidson et al. (Proc. Natl. Acad. Sci. USA 97:3428-3432, 2000), Xiao et al. (J. Virol. 72: 2224-2232, 1998), Halbert et al. (J. Virol. 74: 1524-1532, 2000); Halbert et al. (J. Virol. 75: 6615-6624, 2001), and Auricchio et al. (Hum. Molec. Genet. 10: 3075-3081, 2001), whose publications are incorporated herein by reference with regard to AAV vectors for gene delivery.

[0212] Also useful in conjunction with the compositions and methods described in this document are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV2) pseudotyped with a capsid protein derived from a serotype different from the serotype provided (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, Petition 870250096842, dated 10 / 23 / 2025, page 81 / 180 74 / 121 AAV7, AAV8, or AAV9, among others). For example, a representative pseudotyped vector is an AAV2 vector encoding a pseudotyped therapeutic protein with a capsid protein derived from AAV serotype 8 or AAV serotype 9. In some embodiments, the pseudotyped AAV has the ITRs of one AAV serotype (e.g., AAV2) and the VP1, VP2, and / or VP3 capsid proteins of a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al. (J. Virol. 74: 1524-1532, 2000); Zolotukhin et al. (Methods. 28: 158-167, 2002); and Auricchio et al. (Hum. Molec. Genet. 10: 3075-3081, 2001).

[0213] In some embodiments, the AAV comprises a capsid disclosed, for example, in WO 2017 / 218842, which disclosure is incorporated herein by reference. In some embodiments, the AAV comprises a capsid protein disclosed in Lin et al. (Mol Brain. 13:138, 2020), which disclosure is incorporated herein by reference. In some embodiments, the AAV comprises an AAV2-retro or AAV9-retro capsid protein. In some embodiments, the AAV comprises a capsid protein that is conjugated to a ligand or an aptamer.

[0214] AAV virions that have mutations within the virion capsid can be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations to facilitate AAV targeting to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, is described in Wu et al. (J. Virol. 74: 8635-45, 2000). Other rAAV virions Petition 870250096842, dated 10 / 23 / 2025, page 82 / 180 75 / 121 that can be used in the methods of the invention include those capsid hybrids that are generated by molecular reproduction of viruses as well as by exon shuffling. See, for example, Soong et al. (Nat. Genet. 25: 436-439, 2000) and Kolman and Stemmer (Nat. Biotechnol. 19: 423-428, 2001). iii. Methods for delivering a code-optimized gene product

[0215] In addition to the virus-based administration methods described above, a variety of nonviral techniques can also be used to introduce a codon- or coding sequence-optimized gene into a subject or host cell (e.g., a host cell derived from a human subject). For example, electroporation can be used to permeabilize mammalian cells (e.g., human cells) by applying an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al. (Nucleic Acids Res. 15: 1311, 1987), the disclosure of which is incorporated herein by reference.A similar technique, Nucleofection™, uses an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection™ and useful protocols for performing this technique are described in detail, for example, in Distler et al. (Exp. Dermatol. 14:315, 2005), as well as in US 2010 / 0317114, disclosures of which are incorporated herein by reference.

[0216] Additional useful techniques for target cell transfection include the compression poration methodology. This technique induces rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technology is advantageous insofar as a vector is not required for the delivery of nucleic acids into a cell, such as Petition 870250096842, dated 10 / 23 / 2025, page 83 / 180 76 / 121 a human target cell. Compression poration is described in detail, for example, in Sharei et al. (J. Vis. Exp. 81:e50980, 2013), which is incorporated herein by reference.

[0217] Lipofection represents another useful technique for transfecting target cells. This method involves loading nucleic acids into a liposome, which often features cationic functional groups such as quaternary or protonated amines, toward the exterior of the liposome. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to the uptake of exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Patent No. 7,442,386, disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to induce the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex.Exemplary cationic molecules that associate with polynucleotides to confer a positive charge favorable to interaction with the cell membrane are activated dendrimers (described, for example, in Dennig (Topics in Current Chemistry 228:227, 2003), which is incorporated herein by reference) and diethylaminoethyl (DEAE)dextran, whose use as a transfection agent is described in detail, for example, in Gulick et al. (Curr. Protoc. in Mol. Biol. 40:1:9.2:9.2.1, 1997), which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells gently and efficiently, as this methodology uses an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, for example, in US 2010 / 0227406, which is incorporated herein by reference. Petition 870250096842, dated 10 / 23 / 2025, page 84 / 180 77 / 121

[0218] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al. (Methods in Cell Biology 82:309, 2007), whose publication is incorporated herein by reference.

[0219] Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described in this document. For example, microvesicles induced by co-overexpression of the glycoprotein VSV-G with, for example, a genome-modifying protein such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyzes site-specific cleavage of an endogenous polynucleotide sequence in order to prepare the cell's genome for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also called Gesicles, for the genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Mol. Ther. 23: Supplement 1, Abstract No. 122, 2015). C. Treatment methods i. Therapeutic utility of codon-optimized genetic sequences

[0220] The codon optimization and protein production methods described herein can be applied to methods of treating a disease or condition in a subject who needs them. In some modalities, a treatment method may be prophylactic treatment for a subject at risk of a disease or condition. In other modalities, a treatment method may reduce, reverse, improve, stabilize, or enhance Petition 870250096842, dated 10 / 23 / 2025, page 85 / 180 78 / 121 a disease state or condition in a subject. In other modalities, methods can be used to alleviate, improve, reduce, or reverse clinical manifestations of a disease or condition.

[0221] In some modalities, the treatment method is intended to treat a disease or condition caused by a defect or deficiency in a single gene or single gene product (e.g., a single polynucleotide or protein). In some modalities, the treatment method is intended to treat a disease or condition that leads to a defect or deficiency in multiple gene products. In some modalities, the defect or deficiency is defined by reduced expression, reduced activity, and / or aberrant localization of one or more gene products. In some modalities, the gene product is distributed for transient expression. In other modalities, the gene product integrates into the host genome.

[0222] In some modalities, the treatment method is intended to replace, supplement, or replenish a missing, deficient (e.g., low expression levels), or defective (e.g., mutant, loss of function, or low biological or catalytic activity) gene product in a subject with a disorder or condition. In some modalities, the disorder or condition is characterized by a loss-of-function mutation or a gene deletion. In other modalities, the disorder or condition is acquired (e.g., a deficient or defective gene product from stochastic or environmental factors). In some modalities, the codon-optimized gene product encodes a polypeptide or protein that is identical (i.e., retains 100% sequence identity) to the wild-type amino acid sequence to replace, supplement, or replenish low levels of one or more deficient or defective gene products.In other embodiments, the codon-optimized gene product encodes a polypeptide or protein that shares at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, ...). Petition 870250096842, dated 10 / 23 / 2025, page 86 / 180 79 / 121 96%, 97%, 98%, or 99% sequence identity with the wild-type amino acid sequence to encode a polypeptide or protein that has enhanced biological function (e.g., increased catalytic function or reduced immunogenicity).Some examples in which a polypeptide or protein can be modified to improve biological function include mutation or addition of sites for post-translational modifications (e.g., glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, among others, or a combination thereof), mutation or addition of cysteines for altered or added disulfide bonds, modification of binding sites for enhanced binding activity between the protein of interest and one or more known binding partners, modification of protease binding sites or targeted cleavage, modification of a signal sequence for enhanced secretion or altered localization, among other protein modifications known in the art.

[0223] In some modalities, the treatment method is intended to increase or supplement the expression of a normally expressed gene product (e.g., present at a concentration within an accepted healthy range) in a subject with a disorder or condition that would benefit from increased expression of said gene product. Increased expression of a normally expressed gene product may be desired to increase the rate of an enzymatic reaction, increase the potency or rate of a signaling response (e.g., intracellularly or extracellularly), modulate the trafficking or adhesion of a cell or cellular component, increase the probability or propensity for binding or occurrence of a transient interaction (e.g., based on the affinity or Kd of two or more molecules), or otherwise modulate one or more biological processes.Such methods can be clinically useful for increasing the expression of a protein with redundant function to replace a deficient or defective protein. Petition 870250096842, dated 10 / 23 / 2025, page 87 / 180 80 / 121 Such methods may also be clinically useful for modulating a disease-causing gene product that is logistically more difficult to employ as a codon-optimized gene product for a treatment method due to a variety of non-limiting factors, including large gene size, low accessibility to a target cell or tissue, and / or high immunogenicity of the gene product.

[0224] In some modalities, the treatment method increases the expression of a protein or polypeptide (e.g., increases, induces the expression of an exogenous protein or polypeptide) to modulate or regulate a separate causative agent underlying a disease. Such modalities may be clinically useful for blocking, inhibiting, proteolyzing, mediating the clearance of, or otherwise attenuating the effect of a causative agent, such as a pathogen (e.g., a virus, a bacterium, a fungus, or a parasite) or a pro-inflammatory protein (e.g., a cytokine or a cytokine receptor).

[0225] In some embodiments, treatment with an effective amount of a codon-optimized gene product (e.g., a polynucleotide encoding a protein of interest; e.g., a transgene packaged in an AAV to encode a protein of interest) or a pharmaceutical composition containing the same increases gene product expression by about 5% to 50% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50%), by about 50% to 100% (about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or approximately 100%), or more than 100% (approximately 100%, approximately 125%, approximately 150%, approximately 175%, approximately 200%, approximately 225%, approximately 250%, approximately 275%, approximately 300% or more) compared to a reference sample, such as a Petition 870250096842, dated 10 / 23 / 2025, p. 88 / 18081 / 121 biological sample from a healthy control subject or a biological sample from the same subject before treatment administration. In some modalities, treatment with an effective amount of a codon-optimized gene product (e.g., a polynucleotide encoding a protein of interest; e.g., a transgene packaged in an AAV to encode a protein of interest) increases gene product expression by approximately 1-fold, approximately 2-fold, approximately 3-fold, approximately 4-fold, approximately 5-fold, approximately 6-fold, approximately 7-fold, approximately 8-fold, approximately 9-fold, approximately 10-fold, approximately 11-fold, approximately 12-fold, approximately 13-fold, approximately 14-fold, approximately 15-fold, approximately 16-fold, approximately 17-fold, approximately 18-fold, approximately 19-fold, approximately 20-fold, approximately 25-fold, approximately 30-fold, approximately 35-fold, approximately 40-fold. times, in about 45 times,by approximately 50 times or more compared to a reference sample, such as a biological sample from a healthy control subject or a biological sample from the same subject before treatment administration. In either of the foregoing modalities, treatment with an effective amount of a codon-optimized gene product can result in increased expression globally (e.g., in all organ systems or all tissues of a subject; e.g., throughout the body) or locally (e.g., in a subset of tissues, in one or more organs, or in one or more organ systems of interest).

[0226] In some modalities, the treatment method is directed at treating a metabolic disorder, a blood disorder, a cardiovascular disorder, a neurological disorder, an eye or ophthalmological disorder, a reproductive disorder, an infectious disease, an autoimmune or immunological disorder, or a type of cancer. Exemplary diseases and genes that may benefit from the methods and applications described herein are Petition 870250096842, dated 10 / 23 / 2025, page 89 / 180 82 / 121 summarized in Table 2 below. Table 2: Exemplary Diseases and Target Genes Disease(s) Gene(s) Achondroplasia FGFR3 Achromatopsia CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, ACHM2, ACHM3 Acute Kidney Injury NFkappaB, AATF, p85alpha, FAS, elements of the apoptosis cascade (e.g., FASR, Caspase 2, 3, 4, 6, 7, 8, 9, 10, AKT, TNF alpha, IGF1, IGF1R, RIPK1), p53 Age-Related Macular Degeneration Abcr, CCL2, CC2, CP, Timp3, cathepsin D, VLDLR, CCR2, sFLTOl (VEGF / PlGF (placental growth factor) binding domain of human VEGFRI / Flt-1 (hVEGFRl) fused to the Fe portion of human IgG(l) via a ligand (of polyglycine) Acquired Immunodeficiency Syndrome (AIDS) KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1 Albinism (including oculocutaneous albinism (Types 1-7) and ocular albinism) TYR, OCA2, TYRP1, SLC45A2, SLC24A5, C10orf11 Alkaptonuria HGD Alpha-1 antitrypsin deficiency (AATD or AlAD) or hereditary emphysema AAT, SERPlNA1, those established in WO2017165862, PiZ allele Amyotrophic lateral sclerosis (ALS) SOD1, ALS2, ALS3, ALS5, ALS7, STEX,FUS, TARDBP, VEGF (VEGF-a; VEGF-b; VEGF-c), DPP6, NEFH, PTGS1, SLC1A2, TNFRSF10B, PRPH, HSP90AA1, CRIA2, IFNG, AMPA2 S100B, FGF2, AOX1, CS, TXN, RAPHJ1, MAP3K5, NBEAL1, GPX1, ICA1L, RAC1, MAPT, ITPR2, ALS2CR4, GLS, ALS2CR8, CNTFR, ALS2CR11, FOLH1, FAM117B, P4HB, CNTF, SQSTM1, STRADB, NAIP, NLR, YWHAQ, SLC33A1, TRAK2, SCA1, NIF3L1, NIF3, PARD3B, COX8A, CDK15, HECW1, HECT, C2, WW 15, NOS1, MET, SOD2, HSPB1, NEFL, CTSB, ANG, HSPA8, RNase A, VAPB, VAMP, SNCA, alpha HGF, CAT, ACTB, NEFM, TH, BCL2, FAS, CASP3, CLU, SMN1, G6PD, BAX, HSF1, RNF19A, JUN, ALS2CR12, HSPA5, MAPK14, APEX1, TXNRD1, NOS2, TIMP1, CASP9, XIAP, GLG1, EPO, VEGFA, ELN, GDNF, NFE2L2, SLC6A3, HSPA4, APOE, PSMB8, DCTN2, TIMP3, KIFAP3, SLC1A1, SMN2, CCNC, STUB1, ALS2, PRDX6, SYP, CABIN1, CASP1, GART, CDK5, ATXN3, RTN4, C1QB, VEGFC, HTT, PARK7, XDH, GFAP, MAP2, CYCS, FCGR3B, CCS, UBL5, MMP9m SLC18A3, TRPM7, HSPB2, AKT1, DEERL1, CCL2, NGRN, GSR, TPPP3, APAF1, BTBD10, GLUD1, CXCR4, SLC1A3, FLT1, PON1, AR, LIF, ERBB3, GAS1, CD44,TP53, TLR3, GRIA1, GAPDH, AMPA, GRIK1, DES, CHAT, FLT4, CHMP2B, BAG1, CHRNA4, GSS, BAK1, KDR, Petition 870250096842, dated 10 / 23 / 2025, pp. 90 / 180 83 / 121 Disease(s) Gene(s) GSTP1, OGG1, IL6 Alzheimer's disease E1, CHIP, UCH, UBB, Tau, LRP, PICALM, CLU, PS1, SORL1, CR1, VLDLR, UBA1, UBA3, CHIP28, AQP1, UCHL1, UBA1, APP28, AQP1, UCHL1, UCHLAD, APP, APP, CV1 APOE, AD2, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BDNF, BLMH, BMH, PSEN1, AD3, ALAS2, ABCA1, BIN1, BDNF, BTNL8, C1ORF49, CDH4, CHRNB2, CKLFSF2,CLEC4E,CR1L,CSF3R, CST3, CYP2C, DAPK1, ESR1, FCAR, FCGR3B, FFA2, FGA, GAB2, GALP, GAPDHS, GDNF, GMPB, HP, HTR7, IDE, IF127, IFI, 28, IR, IR, IL, 18 IL8RB, JAG1, KCNJ15, LRP6, MAPT, MARK4, MPHOSPH1, MTHFR, NBN, NCSTN, NIACR2, NGF, NMNAT3, NTM, ORM1, P2RY13, PBEF1, PCK1, PICALM, PLAU, PLXNC11, PRNPPS, EN, PTPRA, 1, PTPRA RALGPS2, RGSL2, SELENBP1, SLC25A37, SORL1, Mitoferrin-1, TF, TFAM, TNF, TNFRSF10C, UBE1C Amyloidosis APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB Neuropathy, CDANemia, PALB, Anemia, CDAN CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7,ASAT Angelman Syndrome UBE3A Anxiety BDNF Arthritis, Rheumatoid Arthritis TNFR:Fc Attention Deficit Hyperactivity Disorder (ADHD) PTCHD1 Autoimmune Lymphoproliferative Syndrome TNFRSF6, APT1, FAS, CD95, ALPS1A Autism, autism spectrum disorders (ASDs), including Asperger's syndrome and a general diagnostic category called pervasive developmental disorders (PDDs) PTCHD1, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, RTT, PPMX, MRX16, RX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR1, FMR2, FXR1, FXR2, MGLUR5, ATP10C, CDH10, GRM6, MGLUR6, CDH9, CNTN4, NLGN2, CNTNAP2, SEMA5A, DHCR7, NLGN4X, NLGN4Y, DPP6, NLGN5, EN2, NRCAM, MDGA2, NRXN1, FMR2, AFF2, FOXP2, OR4M2, OXTR, FXR1, FXR2, PAH, GABRA1, PTEN, GABRA5, PTPRZ1, GABRB3, GABRG1, HIRIP3, SEZ6L2, HOXA1, SHANK3, IL6, SHBZRAP1, LAMB1, SLC6A4, SERT, MAPK3, TAS2R1, MAZ, TSC1, MDGA2, TSC2, MECP2, UBE3A, WNT2,See also 20110023145 Autosomal Dominant Polycystic Kidney Disease (ADPKD) - (includes diseases such as von Rippel-Lindau disease and Tuberous Sclerosis Complex disease) PKD1, PKD2 Autosomal Recessive Polycystic Kidney Disease (ARPKR) PKDH1, Petition 870250096842, dated 10 / 23 / 2025, page 91 / 180 84 / 121 Disease(s) Gene(s) Ataxia-Telangiectasia (also known as Louis Bar syndrome) ATM B-cell Non-Hodgkin Lymphoma BCL7A, BCL7 Bardet-Biedl Syndrome ARL6, BBS1, BBS2, BBS4, BBS5, BBS7, BBS9, BBS10, BBS12, CEP290, INPP5E, LZTFL1, MKKS, MKS1, SDCCAGS, TRIM32, TTC8 Nude Lymphocyte Syndrome TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5 Bartter Syndrome (Types I, II, III, IVA and B, and V) SLC12A1 (type I), KCNJ1 (type II), CLCNKB (type III), BSND (type IV A), or both genes CLCNKA CLCNKB (type IV B), CASR (type V) Batten disease, Late Infantile Neuronal Ceroid Lipofuscinosis CLN2 Becker Muscular Dystrophy DMD, BMD, MYF6 Best disease (Vitelliform Macular Dystrophy Type 2) VMD2 Bipolar Disorder BDNF Bleeding Disorders TBXA2R, P2RX1, P2X1 Blue Cone Monochromacy OPN1LW, OPN1MW, LCR Breast Cancer BRCA1, BRCA2, COX-2 Bruton disease (also known as X-linked Agammaglobulinemia) BTK Canavan disease Cancers ASPA (e.g.,lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic B-cell leukemia (A-ALL), acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin lymphoma (NHL), diffuse large cell lymphoma (DLCL), multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma) FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC, TRBC, those described in WO2015048577 Cardiovascular Diseases IL1B, XDH, TP53, PTGS, MB, IL4, ANGPT1, ABCGu8, CTSK, PTGIR, KCNJ11, INS, CRP, PDGFRB, CCNA2, PDGFB, KCNJ5, KCNN3, CAPN10, ADRA2B, ABCG5, PRDX2, CPAN5, PARP14, MEX3C, ACE, RNF, IL6, TNF, STN, SERPINE1, ALB, ADIPOQ, APOB, APOE, LEP, MTHFR, APOA1, EDN1, NPPB, NOS3, PPARG, PLAT, PTGS2, CETP, AGTR1, HMGCR, IGF1, SELE, REN, PPARA, PON1, KNG1, CCL2, LPL, VWF, F2, ICAM1, TGFB, NPPA,IL10, EPO, SOD1, VCAM1, IFNG, LPA, MPO, ESR1, MAPK, HP, F3, CST3, COG2, MMP9, SERPINC1, F8, HMOX1, APOC3, IL8, PROL1, CBS, NOS2, TLR4, SELP, ABCA1, AGT, LDLR, GPT, VEGFA, NR3C2, IL18, NOS1, NR3C1, FGB, HGF, IL1A, Petition: 870250096842, on 10 / 23 / 2025, p. 92 / 180 85 / 121 Subject Gene(s) AKT1, LIPC, HSPD1, MAPK14, SPP1, ITGB3, CAT, UTS2, THBD, F10, CP, TNFRSF11B, EGFR, MMP2, PLG, NPY, RHOD, MAPK8, MYC, FN1, CMA1, PLAU, GNB3, ADRB2, SOD2, F5, VDR, ALOX5, HLA- DRB1, PARP1, CD40LG, PON2, AGER, IRS1, PTGS1, ECE1, F7, IRMN, EPHX2, IGFBP1, MAPK10, FAS, ABCB1, JUN, IGFBP3, CD14, PDE5A, AGTR2, CD40, LCAT, CCR5, MMP1, TIMP1, ADM, DYT10, STAT3, MMP3, ELN, USF1, CFH, HSPA4, MMP12, MME, F2R, CELL, CTSB, ANXA5, ADRB1, CYBA, FGA, GGT1, LIPG, HIF1A, CXCR4, PROC, SCARB1, CD79A, PLTP, ADD1, FGG, SAA1, KCNH2, DPP4, NPR1, VTN, KIAA0101, FOS, TLR2, PPIG, IL1R1, AR, CYP1A1, SERPIN1, MTR, RBP4, APOA4, CDKN2A, FGF2, EDNRB, ITGA2, VLA-2, CABIN1, SHBG, HMGB1, HSP90B2P, CYP3A4, GJA1, CAV1, ESR2, LTA, GDF15, BDNF, CYP2D6, NGF, SP1, TGIF1, SRC, EGF, PIK3CG, HLA-A, KCNQ1, CNR1, FBN1, CHKA, BEST1, CTNNB1, IL2, CD36, PRKAB1, TPO, . ALDH7A1, CX3CR1, TH, F9, CH1, TF, HFE, IL17A, PTEN, GSTM1, DMD, GATA4, F13A1, TTR, FABP4, PON3, APOC1, INSR, TNFRSF1B, HTR2A, CSF3, CYP2C9, TXN, CYP11B2, .PTH, CSF2, KDR, PLA2G2A, THBS1, GCG, RHOA, ALDH2, TCF7L2, NFE2L2, NOTCH1, UGT1A1, IFNA1, PPARD, SIRT1, GNHR1, PAPPA, ARR3, NPPC, AHSP, PTK2, IL13, MTOR, ITGB2, GSTT1, IL6ST, CPB2, CYP1A2, HNF4A, SLC64A, PLA2G6, TNFSF11, SLC8A1, F2RL1, AKR1A1, ALDH9A1, BGLAP, MTTP, MTRR, SULT1A3, RAGE, C4B, P2RY12, RNLS, CREB1, POMC, RAC1, LMNA, CD59, SCM5A, CYP1B1, MIF, MMP13, TIMP2, CYP19A1, CUP21A2, PTPN22, MYH14, MBL2, SELPLG, AOC3, CTSL1, PCNA, IGF2, ITGB1, CAST, CXCL12, IGHE, KCNE1, TFRC, COL1A1, COL1A2, IL2RB, PLA2G10, ANGPT2, PROCR, NOX4, HAMP, PTPN11, SLCA1, IL2RA, CCL5, IRF1, CFAR, CACA, EIF4E, GSTP1, JAK2, CYP3A5, HSPG2, CCL3, MYDSS, VIP, SOAT1, ADRBK1, NR4A2, MMPS, NPR2, GCH1, EPRS, PPARGC1A, F12, PECAM1, CCL4, CERPINA34, CASR, FABP2, TTF2, PROS1, CTF1, SGCB, YME1L1, CAMP, ZC3H12A, AKR1B1, MMP7, AHR, CSF1, HDAC9, CTGF, KCNMA1, UGT1A, PRKCA, COMT, S100B, EGR1, PRL, IL15, DRD4, CAMK2G, SLC22A2, CCL11, PGF, THPO, GP6, TACR1, NTS, HNF1A, SST, KCDN1, LOC646627, TBXAS1, CUP2J2, TBXA2R, ADH1C,ALOX12, AHSG, BHMT, GJA4, SLC25A4, ACLY, ALOX5AP, NUMA1, CYP27B1, CYSLTR2, SOD3, LTC4S, UCN, GHRL, APOC2, CLEC4A, KBTBD10, TNC, TYMS, SHC1, LHC13, KSOK3, ADHK3, KSO HSD11B1, VKORC1, SERPINB2, TNS1, Petition 870250096842, of 23 / 10 / 2025, p. 93 / 180 86 / 121 Doença(s) Gene(s) RNF19A, EPOR, ITGAM, PITX2, MAPK7, FCGR3A, LEEPR, ENG, GPX1, GOT2, HRH1, NR112, CRH, HTR1A, VDAC1, HPSE, SFTPD, TAP2, RMF123, PTK2Bm NTRK2, IL6R, ACHE, GLP1R, GHR, GSR, NQO1, NR5A1, GJB2, SLC9A1, MAOA, PCSK9, FCGR2A, SERPINF1, EDN3, UCP2, TFAP2A, C4BPA, SERPINF2, TYMP, ALPP, CXCR2, SLC3A3, ABCG2, ADA, JAK3, HSPA1A, FASN, FGF1, F11, ATP7A, CR1 GFPA, ROCK1, MECP2, MYLK, BCHE, LIPE, ADORA1, WRN, CXCR3, CD81, SMAD7, LAMC2, MAP3K5, CHGA, IAPP, RHO, ENPP1, PTHLH, NRG1, VEGFC, ENPEP, CEBPB, NAGLU, F2RL3, CX3CL1, BDKRB1, ADAMTS13, ELANE, ENPP2, CISH, GAST, MYOC, ATP1A2, NF1, GJB1, MEF2A, VCL, BMPR2, TUBB, CDC42, KRT18, HSF1, MYB, PRKAA2, ROCK2, TFP1, PRKG1, BMP2, CTNND1, CTH, CTSS, VAV2, NPY2R, IGFBP2, CD28, GSTA1 PPIA, APOH, S100A8, IL11, ALOX15, FBLN1, NR1H3, SCD, GIP, CHGB, PRKCB, SRD5A1, HSD11B2, CALCRL, GALNT2, ANGPTL4, KCNN4, PIK3C2A, HBEGF, CYP7A1, HLA-DRB5, BNIP3, GCKR, S100A12, PADI4, HSPA14, CXCR1, H19, KRTAP19-3, IDDM2, RAC2, YRY1, CLOCK, NGFR, DBH, CHRNA4,CACNA1C, PRKAG2, CHAT, PTGDS, NR1H2, TEK, VEGFB, MEF2C, MAPKAPK2, TNFRSF11A, HSPA9, CYSLTR1, MAT1A, OPRL1, IMPA1, CLCN2, DLD, PSMA6, PSMBS, CHI3L1, ALDH1B1, PARP2, STAR, LBP, ABCC6, RGS2, EFNB2, GJB6, APOA2, AMPD1, DYSF, FDFT1, EMD2, CCR6, GJB3, IL1RL1, ENTPD1, ​​BBS4, CELSR2, F11R, RAPGEF3, HYAL1, ZNF259, ATOX1, ATF6, KHK, SAT1, GGH, TIMP4, SLC4A4, PDE2A, PDE3B, FADS1, FADS2, TMSB4X, TXNIP, LIMS1, RHOB, LY96, FOXO1, PNPLA2, TRH, GJC1, SLC7A5, FTO, GJD2, PRSC1, CASP12, GPBAR1, PXK, IL33, TRIB1, PBX4, NUPR1, 15SEP, CILP2, TERC, GGT2, MTCO1, UOX, AVP, ANGPLT3 Cataract CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CCL, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM,KRIT1 Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) CASQ2 CDKL-5 Deficiencies or CDKL5-Mediated Diseases Charcot-Marie-Tooth Disease (CMT) (types 1, 2, 3 and 4) PMP22 (CMT1A and E), MPZ (CMT1B), LITAF (CMT1C), EGR2 (CMT1D), NEFL (CMT1F), Petition 870250096842, dated 10 / 23 / 2025, p. 94 / 180 87 / 121 Disease Gene(s) GJB1 (CMT1X), MFN2 (CMT2A), KIF1B (CMT2A2B), RAB7A (CMT2B), TRPV4 (CMT2C), GARS (CMT2D), NEFL (CMT2E), GAPD1 (CMT2K), HSPB8 (CMT2L), DYNC1H1 (CMT2O), LRSAM1 (CMT2P), IGHMBP2 (CMT2S), MORC2 (CMT2Z), GDAP1 (CMT4A), MTMR2 or SBF2 / MTMR13 (CMT4B), SH3TC2 (CMT4C), NDRG1 (CMT4D), PRX (CMT4F), FIG4 (CMT4J), NT-3 Chediak-Higashi Syndrome LYST Choroideremia CHM, REP1 Atrophy Chorioretinal PRDM13, RGR, TEAD1 Chronic Granulomatous Disease CYBA, CYBB, NCF1, NCF2, NCF4 Chronic Heart Failure SERCA2 Chronic Mucocutaneous Candidiasis AIRE, CARD9, CLEC7A IL12B, IL12B1, IL1F, IL17RA, IL17RC, RORC, STAT1, STAT3, TRAF31P2 Cirrhosis KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988 Colon Cancer (Familial Adenomatous Polyposis (FAP) and Hereditary Nonpolyposis Colorectal Cancer (HNPCC)) FAP:APC, HNPCC: MSH2, MLH1, PMS2, SH6, PMS1 Combined Immunodeficiency IL2RG, SCIDX1, SCIDX, IMD4, HIV-1 (CCL5, SCYA5, D17S136E, TCP228) Cone(-rod) dystrophy AIPL1, CRX, GUA1A, GUCY2D, PITPM3, PROM1,PRPH2, RIMS1, SEMA4A, ABCA4, ADAM9, ATF6, C21ORF2, C8ORF37, CACNA2D4, CDHR1, CERKL, CNGA3, CNGB3, CNNM4, CNAT2, IFT81, KCNV2, PDE6C, PDE6H, POC1B, RAX2, RDH5, RPGRIP1, TTLL5, RetCG1, GUCY2E Congenital Stationary Night Blindness CABP4, CACNA1F, CACNA2D4, GNAT1, CPR179, GRK1, GRM6, LRIT3, NYX, PDE6B, RDH5, RHO, RLBP1, RPE65, SAG, SLC24A1, TRPM1 Congenital Fructose Intolerance ALDOB Cori Disease (Glycogen Storage Disease) Type III) AGL Corneal opacity and dystrophy APOA1, TGFB1, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD Congenital flat cornea KERA, CNA2 Cri du Chat syndrome, also known as 5p syndrome and Cat Cry Syndrome Deletions involving only the 5p15.2 band along the entire short arm of chromosome 5, for example, CTNND2, TERT Crigler-Najjar syndrome UGT1A1 Cystic fibrosis (CF) CTFR, ABCC7, CF, MRP7, SCNN1A, those described in WO2015157070 Diabetic nephropathy Gremlin,12 / 15- lipoxygenase, TIM44 Dental disease (types 1 and 2) Type 1: CLCN5, Type 2: ORCL, Petition 870250096842, dated 10 / 23 / 2025, p. 95 / 180 88 / 121 Disease(s) Gene(s) Dentatorrubro-Palidoluysian Atrophy (DRPLA) (also known as Haw River and Naito-Oyanagi Disease) Atrophin-1 and ATN1 Down Syndrome Trisomy 21 Drug Dependence PRKCE, DRD2, DRD4, ABAT, GRIA2, GRM5, GRIN1, HTR1b, GRIN2A, DRD3, PDYN, GRIA1 Duane Syndrome (Types 1, 2 and 3, including subgroups A, B and C). Other names for this condition include Duane Retraction Syndrome (or DR Syndrome), Ocular Retraction Syndrome, Retraction Syndrome, Congenital Retraction Syndrome, and Stilling-Turk-Duane Syndrome CHN1, indels on chromosomes 4 and 8 Duchenne Muscular Dystrophy (DMD) DMD, BMD, dystrophin gene, intron flanking exon 51 of the DMD gene, exon 51 mutations in the DMD gene, see also WO2013163628 and Pub. Pt. US 20130145487 Eating disorder BDNF Edwards Syndrome (Trisomy 18) Complete or partial trisomy of chromosome 18 Ehlers-Danlos Syndrome (Types I-VI) COL5A1, COL5A2, COL1A1, COL3A1, TNXB, PLOD1, COL1A2, FKBP14,ADAMTS2 Emery-Dreifuss Muscular Dystrophy LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A Enhanced Cone Syndrome NR2E3, NRL Epilepsy NPY Fabry Disease GLA, AGA Facioscapulohumeral Muscular Dystrophy FSHMD1A, FSHD1A, FRG1 Factor H and Factor H1 HF1, CFH, HUS Factor V Leiden Thrombophilia and Factor V Deficiency Factor V (F5) Factor V and Factor VII Deficiency MCFD2 Factor VII Deficiency F7 Factor X Deficiency F10 Factor XI Deficiency F11 Factor XII Deficiency F12, HAF Factor XIIIA Deficiency F13A1, F13A Factor XIIIB Deficiency F13B Familial Hypercholesterolemia APOB, LDLR, PCSK9 Familial Mediterranean Fever (FMF), also called Recurrent Polyserositis or Familial Paroxysmal Polyserositis MEFV Fanconi Anemia FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCC, FANCG, RAD51, BRCA1, BRCA2, BRIP1, BACH1, FANCJ, FANCB, FANCD1, FANCD2, FANCD, FAD, FANCE, FACE, FANCF, FANCI, ERCC4, FANCL, FANCM, PALB2, RAD51C, SLX4,UBE2T, FANCB, XRCC9, PHF9, KIAA1596, Petition 870250096842, dated 10 / 23 / 2025, page 96 / 180 89 / 121 Disease(s) Gene(s) Fanconi Syndrome Types I (Childhood onset) and II (Adult-onset) FRTS1, GATM Fragile X Syndrome and related disorders FMR1, FMR2, FXR1, FXR2, mGLUR5 Mental Retardation Fragile XE (also known as Martin Bell Syndrome) FMR1 Friedreich's Ataxia (FRDA) FXN / X25 Fuchs' Corneal Endothelial Dystrophy TCF4, COL8A2 Galactosemia GALT, GALK1, GALE Gastrointestinal epithelial cancer, GI cancer CISH Gaucher Disease (types 1, 2 and 3, as well as other uncommon forms that may not fit into these types) GBA, GCase Glaucoma MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A, those described in WO2015153780 Glomerulosclerosis Chemokine ligand 2 CC Glycogen storage diseases (types I-VI - see also Cori disease, Pompe disease, McArdle disease, Hers disease and Von Gierke disease) SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM,See also Cori disease, Pompe disease, McArdle disease, Hers disease, and Von Gierke disease. Erythrocyte glycolytic enzyme deficiency: any mutations in a gene for an enzyme in the glycolysis pathway, including mutations in genes for hexokinases I and II, glucokinase, phosphoglucose isomerase, phosphofructokinase, aldolase Bm triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase, phosphoglycerate mutase, enolase I, pyruvate kinase. GM2 gangliosidoses (e.g., Sandhoff disease and Tay-Sachs disease): Hexosaminidase-alpha, hexosaminidase-beta, HEX-A. Glycogen storage diseases type Ia (GSDIa): G6Pase. Hemophilia (A or B): F8, F9. Hartnup disease: SLC6A19. Hearing loss. NOX3, Hes5, BDNF Hemochromatosis (HH) HFE, H63D Hemophagocytic Lymphohistiocytosis Disorders PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3 Hemorrhagic Disorders PI, ATT,F5 Hers Disease (Glycogen Storage Disease Type VI) PYGL Hereditary Angioedema (HAE) Kallikrein B1 Hereditary Hemorrhagic Telangiectasia (Osler-Weber-Rendu Syndrome) ACVRL1, ENG, SMAD4 Hereditary Spherocytosis NK1, EPB42, SLC4A1, SPTA1, SPTB Hereditary Persistence of Fetal Hemoglobin HBG1, HBG2, BCL11A, HBG 1 and / or 2 promoter region (in the CCAAT box), Petition 870250096842, dated 10 / 23 / 2025, page 97 / 180 90 / 121 Disease(s) Gene(s) Hemophilia (Hemophilia A (Classic), B (also known as Natal disease) and C) A: FVIII, F8C, HEMA B: FVIX, HEME, FIX C: F9, F11 Hepatic Adenoma TCF1, HNF1A, MODY3 Liver failure, early onset and neurological disorder SCOD1, SCO1 Hepatic Lipase Deficiency LIPC Hepatoblastoma, Cancer and Carcinomas CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPR1, MET, CASP8, MCH5 Hermansky-Pudlak Syndrome HPS1, HPS3, HPS4, HPS5, HPS6, HPS7, DTNBP1, BLOC1, BLOC1S2, BLOC3 Susceptibility to or infection with human immunodeficiency virus (HIV) IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), those in WO2015148670A1 Holoprosencephaly (HPE) (Alobar, Semilobar and Lobar) ACVRL1, ENG, SMAD4 Homocystinuria CBS, MTHFR, MTR, MTRR, MMADHC Human papillomavirus (HPV) HPV16, HPV18 E6 / E7 Herpes simplex virus (HSV)1, HSV2 and related keratitis HSV1 genes (immediate early and late HSV-1 genes (UL1, 1, 5, 5, 6, 8, 9, 12, 15, 16, 18, 19, 22, 23, 26,26.5, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 42, 48, 49.5, 50, 52, 54, S6, RL2, RS1, those described in WO2015153789, WO2015153791 Hunter Syndrome (also known as Mucopolysaccharidosis Type II) IDS Huntington's Disease (HD) and HD-like disorders HD, HTT, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, PRKCE, BDNF, IGF1, EP300, RCOR1, PRKCZ, HDAC4, TGM2, those described in WO2013130824, WO2015089354 Hurler Syndrome (also known as Mucopolysaccharidosis (MPS) Type IH, MPS IH) IDUA, α-L-iduronidase Hurler Syndrome (also known as Mucopolysaccharidosis Type IH-S, MPS IH-S) IDUA, α-L-iduronidase Hyaluronidase deficiency (also known as MPS IX) HYAL1 Hypercholesterolemia or Phenylketonuria (PKU) PAH, LDLR Hyper IgM CD40L Syndrome Hypertension caused kidney damage Mineral corticosteroid receptor Immunodeficiencies CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX,TNFRSF14B, TACI Inborn errors of metabolism: including urea cycle disorders, organic acidemias, fatty acid oxidation defects, aminoacidopathies, carbohydrate disorders, mitochondrial disorders See also: Carbohydrate metabolism disorders (e.g., galactosemia), Amino acid metabolism disorders (e.g., phenylketonuria), Fatty acid metabolism (e.g., MCAD deficiency) Petition 870250096842, dated 10 / 23 / 2025, p. 98 / 180 91 / 121 Disease(s) Gene(s) Urea cycle disorders (e.g., citrullinemia), Organic acidemias (e.g., maple syrup urine disease), Mitochondrial disorders (e.g., MELAS), Peroxisomal disorders (e.g., Zellweger syndrome) Inflammation IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), IL-23, Cx3crl, ptpn22, TNFα, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3c11 Inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease) NOD2, IRGM, LRRK2, ATG5, ATG16L1, IRGM, GATM, ECM1, CDH1, LAMB1, HNF4A, GNA12, IL10, CARD9 / 15, CCR6, IL2RA, MST1, TNFSF15, REL, STAT3, IL23R, IL12B, FUT2 Interstitial Renal Fibrosis TGF-β type II receptor Job's syndrome (also known as Hyper IgE syndrome) STAT3, DOCK8 Juvenile Retinoschisis RS1, XLRS1 Kabuki syndrome 1 MLL4,KMT2D Kennedy disease (also known as Spinobulbar Muscular Atrophy) SBMA / SMAX1 / AR Klinefelter syndrome Extra X chromosome in males Lafora disease EMP2A, EMP2B Leber Congenital Amaurosis CRB1, RP12, CORD2, CRD, CRX, IMPDH1, OTX2, AIPL1, CABP4, CCT2, CEP290, CLUAP1, CRB1, CRX, DTHD1, GDF6, GUCY2D, IFT140, IQCB1, KCNJ13, LCA5, LRAT, NMNAT1, PRPH2, RD3, RDH12, RPE65, RP20, RPGRIP1, SPATA7, TULP1, LCA1, LCA4, GUC2D, CORD6, LCA3 Leber Hereditary Optics ND4 Lesch-Nyhan syndrome HPRT1 Leukocyte deficiencies and disorders ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4 Leukemia TAL1, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH,ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN Shoulder girdle muscular dystrophies LGMD Lipoprotein lipase (LPL) deficiency LPL, Petition 870250096842, dated 10 / 23 / 2025, page 99 / 180 92 / 121 Disease(s) Gene(s) Lowe Syndrome OCRL Lupus Glomerulonephritis MAPK1 Lysosomal Storage Disorders GDNF Machado-Joseph Disease (also known as Spinocerebellar Ataxia Type 3) ATX3 Macular Degeneration ABC4, CBC1, CHM1, APOE, C1QTNF5, C2, C3, CCL2, CCR2, CD36, CFB, CFH, CFHR1, CFHR3, CNGB3, CP, CRP, CST3, CTSD, CX3CR1, ELOVL4, ERCC6, FBLN5, FBLN6, FSCN2, HMCN1, HTRA1, IL6, IL8, PLEKHA1, PROM1, PRPH2, RPGR, SERPING1, TCOF1, TIMP3, TLR3 Macular Dystrophy BEST1, C1QTNF5, CTNNA1, EFEMP1, ELOVL4, FSCN2, GUCA1B, HMCN1, IMPG1, OTX2, PRDM13, PROM1, PRPH2, RP1L1, TIMP3, ABCA4, CFH, DRAM2, IMG1, MFSD8, ADMD, STGD2, STGD3, RDS, RP7, PRPH, AVMD, AOFMD, VMD2 Malattia Leventinese EFEMP1, FBLN3 Malignant Melanoma CD86, B7-2, IL-12 Maple Syrup Urine Disease BCKDHA, BCKDHB,DBT Marfan Syndrome FBN1 Maroteaux-Lamy Syndrome (also known as MPS VI) ARSB Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency MCAD McArdle Disease (Glycogen Storage Disease Type V, myophosphorylase deficiency) PYGM Polycystic kidney disease UMOD, HNFJ, FJHN, MCKD2, ADMCKD2 Metachromatic Leukodystrophy ARSA Methylmalonic acidemia (MMA) MMAA, MMAB, MUT, MMACHC, MMADHC, LMBRD1 Mitochondrial heteroplasmy, myoclonic epilepsy with irregular red fibers (MERRF) or Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) APALI Morquio Syndrome (also known as MPS IVA and B) GALNS Mucopolysaccharidosis Type VII (MPSVII) or Sly-GUSB Syndrome Mucopolysaccharidosis diseases (Types IH / S, IH, II, III AB and C, IS, IVA and B, IX, VII and VI) See also Hurler / Scheie Syndrome, Hurler Disease, Sanfilippo Syndrome, Scheie Syndrome, Morquio Syndrome, Hyaluronidase Deficiency,Sly Syndrome and Maroteaux-Lamy Syndrome Muscular Atrophy VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, Petition 870250096842, dated 10 / 23 / 2025, pages 100 / 180 93 / 121 Disease Gene(s) CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1 Muscular Dystrophy Sarcoglycan α, β, γ, Δ, ε or ζ (SGCA, SGCB, SGCG, SGCD, SGCE or SGCZ), FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, LGMD2C, DMDA1, SCG3, ADL, DAG2, LGMD2D, DMDA2, LGMD2E, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, dystrophin, mini-dystrophin, Myotonic Dystrophy (Type 1 and Type 2), CNBP (Type 2), DMPK (Type 1), Neoplasia, PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, NOTCH1, NOTCH2, NOTCH3, NOTCH4, AKT, AKT2, AKT3, HIF, HIF1A, HIF3A, MET, HRG, BCL2, PPAR alpha, PPAR gamma, WT1 (Wilms tumor), FGF receptor family members (5 members: 1, 2, 3, 4, 5), CDKN2A, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (androgen receptor), TSG101, IGF, IGF receptor, IGF1 (4 variants), IGF2 (3 variants), IGF1 receptor,IGF-2 receptor, BAX, BCL2, caspase family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), KRAS, APC Neurofibromatosis (NF) (NF1, formerly Recklinghausen's NF and NF2) NF1, NF2 Niemann-Pick Lipidosis (Types A, B, and C) Types A and B: SMPD1, Type C: NPC1 or NPC2 Noonan Syndrome PTPN11, SOS1, RAF1, KRAS Norrie Disease or X-linked Familial Exudative Vitreoretinopathy NDP North Carolina Macular Dystrophy MCDR1 Ornithine carbamoyltransferase deficiency OTC Osteogenesis Imperfecta (OI) (Types I, II, III, IV, V, VI, and VII) COL1A1, COL1A2, CRTAP, P3H Osteopetrosis LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1 Patau syndrome (Trisomv 13) Additional copy of chromosome 13 Parkinson's disease (PD) SNCA (PARK1), UCHL1 (PARK 5), and LRRK2 (PARK8)NPY Retinal pigment epithelium (RPE) pattern dystrophy RDS / periphery Phenylketonuria (PKU) PAH, PKU1, QDPR, DHPR, PTS, Petition 870250096842, dated 10 / 23 / 2025, pp. 101 / 180 94 / 121 Disease(s) Gene(s) Polycystic Kidney and Liver Disease FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63 Pompe disease GAA Porphyria (actually refers to a group of different diseases, all with a specific abnormality in the heme production process) ALAD, ALAS2, CPOX, FECH, HMBS, PPOX, UROD, UROS Posterior Polymorphic Corneal Dystrophy TCF4, COL8A2 Prader-Willi Syndrome Short arm region deletion of chromosome 15, including UBE3A Primary hyperoxaluria (e.g., type 1) LDHA (lactate dehydrogenase A) and hydroxyacid oxidase 1 (HAO1) ​​Primary open-angle glaucoma (POAG) MYOC Sclerosing cholangitis Primary TCF4, COL8A2 Progeria (also called Hutchinson-Gilford Progeria Syndrome) LMNA Progressive external ophthalmoplegia ANT-I Propionic acidemia PCCA Prostate cancer HOXB13, MSMB, GPRC6A, TP53, GM-CSF Pyruvate dehydrogenase deficiency PDHA1 Kidney / renal carcinoma RLIP76, VEGF Rett syndrome MECP2, RTT, PPMX, MRX16, MRX79,CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x- Synuclein, DJ-1 Retinitis pigmentosa (RP) ADIPOR1, ABCA4, AGBL5, ARHGEF18, ARL2BP, ARL3, ARL6, BEST1, BBS1, BBS2, C2ORF71, C8ORF37, CA4, CERKL, CLRN1, CNGA1, CMGB1, CRB1, CRX, CYP4V2, DHDDS, DHX38, EMC1, EYS, FAM161A, FSCN2, GPR125, GUCA1B, HK1, HPRPF3, HGSNAT, IDH3B, IMPDH1, IMPG2, IFT140, IFTl72, KLHL7, KIAA1549, KIZ, LRAT, MAK, MERTK, MVK, NEK2, NUROD1, NR2E3, NRL, OFD1, PDE6A, PDE6B, PDE6G, POMGNT1, PRCD, PROM1, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, PRPH2, RPB3, RDH12, REEP6, RP39, RGR, RHO, RLBP1, ROM1, RP1, RP1L1, RPY, RP2, RP9, RPE65, RPGR, SAMD11, SAG, SEMA4A, SLC7A14, SNRNP200, SPP2, SPATA7, TRNT1, TOPORS, TTC8, TULP1, USH2A, ZNF408, ZNF513, see also 20120204282 Sanfilippo Syndrome or Mucopolysaccharidosis Type III B (MPS IIIB) NAGLU deficiency of short-chain acyl-CoA dehydrogenase (SCAD) SCAD Scheie syndrome (also known as Mucopolysaccharidosis Type IS (MPS IS)) IDUA,α-L-iduronidase Schizophrenia Neuregulin1 (NRG1), ERB4 (receptor for, Petition 870250096842, dated 10 / 23 / 2025, page 102 / 180 95 / 121 Domain Gene(s) Neuregulin), Complexin1 (CPLX1), TPH1 Tryptophan hydroxylase, TPH2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1), TCF4, COL8A2 Secretase-related disturbances APH-1 (alpha and beta), PSEN1, NCSTN, PEN-2, Nos1, Parp1, Nat1, Nat2, CTSB, APP, APH1B, PSEN2, PSENEN, BACE1, ITM2B, CTSD, NOTCH1, TNF, INS, DYT10, ADAM17, APOE, ACE, STN, TP53, IL6, NGFR, IL1B, ACHE, CTNNB1, IGF1, IFNG, NRG1, CASP3, MAPK1, CDH1, APBB1, HMGCR, CREB1, PTGS2, HES1, CAT, TGFB1, ENO2, ERBB4, TRAPPC10, MAOB, NGF, MMP12, JAG1, CD40LG, PPARG, FGF2, LRP1, NOTCH4, MAPK8, PREP, NOTCH3, PRNP, CTSG, EGF, REN, CD44, SELP, GHR, ADCYAP1, INSR, GFAP, MMP3, MAPK10, SP1, MYC, CTSE, PPARA, JUN, TIMP1, IL5, IL1A, MMP9, HTR4, HSPG2, KRAS, CYCS, SMG1, IL1R1, PROK1, MAPK3, NTRK1, IL13, MME, TKT, CXCR2, CHRM1, ATXN1, PAWR, NOTCJ2, M6PR, CYP46A1, CSNK1D, MAPK14, PRG2, PRKCA, L1 CAM, CD40, NR1I2, JAG2, CTNND1, CMA1, SORT1, DLK1, THEM4,JUP, CD46, CCL11, CAV3, RNASE3, HSPA8, CASP9, CYP3A4, CCR3, TFAP2A, SCP2, CDK4, JOF1A, TCF7L2, B3GALTL, MDM2, RELA, CASP7, IDE, FANP4, CASK, ADCYAP1R1, ATF4, PDGFA, C21ORF33, SCG5, RMF123, NKFB1, ERBB2, CAV1, MMP7, TGFA,RXRA, STX1A, PSMC4, P2RY2, TNFRSF21, DLG1, NUMBL, SPN, PLSCR1, UBQLN2, UBQLN1, PCSK7, SPON1, SILV, QPCT, HESS, GCC1 Selective IgA deficiency Type 1: MSH5, Type 2: TNFRSF13B Severe Combined Immunodeficiency (SCID), SCID-Xl and Adenosine Deaminase (ADA) SCID JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4, those identified in Pub. Ped. Pat. US 20110225664, 20110091441, 20100229252, 20090271881 and 20090222937 Sickle Cell Disease HBB, BCL11A, BCL11Ae, cisregulatory elements of the B-globin locus, HBG 1 / 2 promoter, distal region of the HBG CCAAT box between 92 and -130 of the HBG transcription start site, those described in WO2015148863, WO 2013 / 126794,Pub. Pat. US 20110182867 Spinocerebellar ataxias (SCA types 1, 2, 3, 6, 7, 8, 12 and 17) ATXN1, ATXN2, ATX3 Sorsby Fundus Dystrophy TIMP3 Stargardt Disease ABCR, ELOVL4, ABCA4, PROM1 Thalassemia (Alpha, Beta, Delta) HBA1, HBA2 (Alpha), HBB (Beta), HBB and HBD (delta), LCRB, BCL11A, BCL11Ae, elements, Petition 870250096842, dated 10 / 23 / 2025, p. 103 / 180 96 / 121 Disease(s) Gene(s) cis-regulatory genes of the B-globin locus, HBG 1 / 2 promoter, as described in WO2015148860, US Pat. Pub. 20110182867, 2015 / 148860 Thymic aplasia (DiGeorge syndrome; 22q11.2 deletion syndrome) Deletion of 30 to 40 genes in the middle of chromosome 22 at a location known as 22q11.2, including TBX1, DGCR8 Transthyretin amyloidosis (ATTR) TTR (transthyretin) Trimethylaminuria FMO3 Trinucleotide repeat disorders (usually) HTT, SBMA / SMAX1 / AR, FXN / X25 ATX3, ATXN1, ATXN2, DMPK, Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP - global instability), VLDLR, Atxn7, Atxn10, FEN1, TNRC6A, PABPN1, JPH3, MED15, ATXN3, TBP, CACNA1A, ATXN80S, PPP2R2B, ATXN7, TNRC6B, TNRC6C, CELF3, MAB21L1, MSH2, TMEM185A, SIX5, CNPY3, RAXE, GNB2, RPL14, ATXN8, ISR, TTR, EP400, GIGYF2, OGG1, STC1, CNDP1, C10ORF2, MAML3, DKC1, PAXIP1, CASK, MAPT, SP1, POLG, AFF2, THBS1, TP53, ESR1, CGGBP1, ABT1, KLK3, PRNP, JUN, KCNN3, BAX, FRAXA, KBTBD10, MBNL1, RAD51, NCOA3, ERDA1, TSC1, COMP, GGLC,RRAD, MSH3, DRD2, CD44, CTCF, CCND1, CLSPN, MEF2A, PTPRU, GAPDH, TRIM22, WT1, AHR, GPX1, TPMT, NDP, ARX, TYR, EGR1, UNG, NUMBL, FABP2, EN2, CRYGC, SRP14, CRYGB, PDCD1, HOXA1, ATXN2L, PMS2, GLA, CBL, FTH1, IL12RB2, OTX2, HOXA5, POLG2, DLX2, AHRR, MANF, RMEM158, see also 20110016540 Triple X Syndrome (XXX) Trisomy of chromosome X Turner Syndrome (XO) Monosomy X Tuberous Sclerosis TSC1, TSC2 Usher Syndrome (Types I, II and III) ABHD12, CDH23, CIB2, CLRN1, DFNB31, GPR98, HARS, MYO7A, PCDH15, USH1C, USH1G, USH2A, USH11A, those described in WO2015134812A1 Velocardiofacial Syndrome (also known as 22q11.2 Deletion Syndrome, DiGeorge Syndrome, Conotruncal Facial Anomaly Syndrome (CTAF), Autosomal Dominant Opitz G / BB Syndrome or Cayler Cardiofacial Syndrome) Many genes are deleted, COM,TBX1 and others are associated with symptoms of short-chain acyl-CoA dehydrogenase (VLCAD) deficiency. VLCAD Von Gierke disease (Glycogen Storage Disease Type I) G6PC, SLC37A4 Von Hippel-Lindau syndrome VHL Von Willebrand disease (Types I, II and III) VWF Wilson disease ATP7B Wiskott-Aldrich syndrome WAS, Petition 870250096842, dated 10 / 23 / 2025, page 104 / 180 97 / 121 Disease(s) Gene(s) Xeroderma pigmentosum POLH X-linked myotubular myopathy MTM1 ii. Administration of a codon-optimized gene product

[0227] The treatment method includes administering a codon-optimized gene product or a pharmaceutical composition containing the same to the subject (e.g., a human being) by any appropriate route of administration (e.g., intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intra-arterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous administration). In some embodiments, the treatment method includes administering a codon-optimized gene product or a pharmaceutical composition containing the same to a subject to produce a therapeutic effect. In some embodiments, the codon-optimized gene product or a pharmaceutical composition containing the same is administered to a subject as monotherapy.In some embodiments, the codon-optimized gene product or a pharmaceutical composition containing it is administered to the subject with one or more additional therapies (e.g., 1, 2, 3, 4, or 5 additional therapeutic agents) as a combination therapy. In some embodiments, combination therapy includes the administration of two or more (e.g., 2, 3, 4, 5, or more than 5) codon-optimized gene products or pharmaceutical compositions containing them to a subject. In some embodiments, combination therapy includes the administration of one or more codon-optimized gene products or pharmaceutical compositions containing them to the subject with one or more additional therapeutic agents or other standard treatment interventions for a specific disease or condition. In some embodiments, combination therapy is administered to the subject to produce an additive or synergistic therapeutic effect.

[0228] The codon-optimized genetic product or a composition Petition 870250096842, dated 10 / 23 / 2025, page 105 / 180 98 / 121 pharmaceutical containing the same can be administered in any appropriate dose. The actual dosage amount of a composition from this disclosure administered to a patient may be determined by physical and physiological factors such as body weight, severity of the condition, prior or concurrent therapeutic interventions, patient idiopathic conditions, and route of administration. Depending on the dosage and route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the subject's response. In some modalities, treatment is used to reduce, delay, and / or inhibit the onset or progression of a disease or condition. In other modalities, methods may be used to alleviate, improve, reduce, or reverse clinical manifestations of a disease or condition. In some modalities, the treatment method includes administering one or more codon-optimized gene products to a subject in a single dose.In some modalities, the treatment method involves administering one or more codon-optimized gene products to a subject in multiple doses. Administration may occur any appropriate number of times per day, week, month, or year, and for as long as necessary. Subjects may be adult or pediatric humans, with or without a comorbid condition. iii. Assessing effectiveness

[0229] Determining the effectiveness of a treatment method described herein may require evaluating the subject's response to treatment. The subject's response may be evaluated in a hospital or outpatient treatment setting. Evaluation of the subject's response may occur one or more times after administration of one or more codon-optimized gene products or a pharmaceutical composition containing them. Evaluation of the subject's response may occur continuously, sporadically, or at designated time points after administration of one or more codon-optimized gene products. Evaluation of the subject's response may occur, for example, 1 to 10 days after the Petition 870250096842, dated 10 / 23 / 2025, pp. 106 / 180 99 / 121 administration of one or more codon-optimized gene products (e.g., 7 to 10 days, 6 to 8 days, 5 to 7 days, 3 to 5 days, 2 to 4 days, 1 to 3 days, or within 1 day after administration of one or more codon-optimized gene products; e.g., 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or less than one day after administration of one or more codon-optimized gene products).The assessment of the subject's response may occur 1 to 12 weeks or more after administration of one or more codon-optimized gene products (e.g., 10 to 12 weeks, 8 to 12 weeks, 6 to 12 weeks, 8 to 10 weeks, 6 to 10 weeks, 4 to 10 weeks, 6 to 8 weeks, 4 to 8 weeks, 2 to 8 weeks, 4 to 6 weeks, 2 to 6 weeks, 3 to 6 weeks, 2 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks after administration of one or more codon-optimized gene products; e.g., later than 12 weeks, 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of one or more codon-optimized gene products). codon).The subject's response can be assessed 1 to 12 months after administration of one or more codon-optimized gene products (e.g., 10 to 12 months, 8 to 10 months, 6 to 8 months, 4 to 6 months, 3 to 5 months, 2 to 4 months, or 1 to 3 months after administration of one or more codon-optimized gene products; e.g., 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month after administration of one or more codon-optimized gene products). The subject's response may be assessed 1 to 5 years or more after administration of one or more codon-optimized gene products (e.g., 4 to 5 years, 3 to 5 years, 2 to 3 years, or 1 to 3 years after administration of one or more codon-optimized gene products; e.g., later than 5 years, 5 years, 4 years, 3 years, 2 years, or 1 year after administration of one or more codon-optimized gene products).

[0230] The effectiveness of the treatment method can be evaluated Petition 870250096842, dated 10 / 23 / 2025, page 107 / 180 100 / 121 comparing one or more metrics of a subject receiving treatment compared to a relevant control or reference. In some modalities, the effectiveness of the treatment method is evaluated by comparing one or more metrics of a subject undergoing treatment with one or more metrics of the same subject before treatment. In some modalities, the effectiveness of the treatment method is evaluated by comparing one or more metrics of a subject undergoing treatment with one or more metrics of a different subject who has the same disease or condition and has not undergone treatment. In other modalities, the effectiveness of the treatment method is evaluated by comparing one or more metrics of a subject undergoing treatment with one or more metrics of a healthy subject (e.g., someone who does not have the disease or condition).

[0231] In some embodiments, the metric is the expression level of the protein or polypeptide encoded by a codon-optimized gene product or an mRNA transcript thereof. In some embodiments, the metric is the biological activity or function of a protein or polypeptide, where the protein is the same as that encoded by the codon-optimized gene product, or a different protein (e.g., a downstream protein, an effector protein, or a binding protein). In some embodiments, the expression or activity of one or more proteins of interest is assessed by collecting one or more biological samples from the subject and comparing the results of previous measurements of the subject and / or results of the subject that were measured before the administration of one or more codon-optimized gene products.In some embodiments, the expression or activity of one or more proteins of interest is detected by biological or analytical methods known in the art or described herein.

[0232] Depending on the effectiveness of the treatment administered to the subject, the treatment regimen may change over the course of treatment. The treatment regimen and its effectiveness can be determined by a qualified professional in the field (e.g., a doctor, clinician, or medical specialist). Petition 870250096842, dated 10 / 23 / 2025, pp. 108 / 180 101 / 121 In some embodiments, the dose of one or more codon-optimized gene products or a pharmaceutical composition containing them may be adjusted (e.g., decreased or increased) over the course of treatment. In some embodiments, the dose of one or more codon-optimized gene products or a pharmaceutical composition containing them may be increased by approximately 10%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200% or more over the course of treatment. In some embodiments, the dose of one or more codon-optimized gene products or a pharmaceutical composition may be decreased by approximately 10%, 25%, 50%, 75% or more than 75% over the course of treatment. In some embodiments, the frequency of administration of one or more codon-optimized gene products or a pharmaceutical composition may increase or decrease over the course of treatment.In other modalities, one or more additional therapies may be added to the treatment regimen, depending on the disease or condition and its treatment pattern. III. Kits

[0233] The compositions or methods described herein may be provided in a kit for use in the production of a protein or gene product. In some embodiments, the compositions and methods described herein may be provided in a kit for use in the treatment of a disease or condition. In some embodiments, the kit may include a package insert instructing the kit user to perform any of the codon optimization methods described herein. In other embodiments, the kit may include a package insert instructing the kit user to perform any of the treatment methods described herein. The kit may optionally include a syringe or device for administering the compositions of this disclosure. In some embodiments, the kit may include one or more additional therapeutic agents. In some embodiments, the kit includes one or more antibodies or binding molecules to detect the Petition 870250096842, dated 10 / 23 / 2025, pp. 109 / 180 102 / 121 expression or activity of a protein of interest. EXAMPLES Example 1. Determination of the yield of the fluorescent protein ZsGreen following 4 distinct codon optimization methods.

[0234] This example describes the codon optimization process of a sequence encoding a ZsGreen fluorescent protein reporter using two common codon usage schemes and comparatively analyzing the resulting sequences and protein production from them with or without applying a series of codon usage rules to reduce or eliminate DRACH motifs. The two common codon usage schemes are the “all-best” scheme (also called “AllBest” in this example), which refers to replacing one or more codons with the synonymous codon with the highest frequency of use, and the “proportional usage” scheme (also called “AllProb” in this example), which refers to modifying the gene or codon sequence so that one or more codons are represented proportionally to their reported frequency of use. Exemplary codons and their frequency of use are shown in Table 1.Codon usage rules (also called "pRules" in this example) are a strategy to reduce m6A modifications by removing DRACH motifs and, in this and subsequent examples, are applied after modifying the encoding sequence with one of the described codon usage schemes. The codon usage rules, or pRules, are as follows: 1. Never use GAC for aspartic acid (D) 2. Never use GAA for glutamic acid (E) 3. Never use GGA for glycine (G) 4. Never use AAA for lysine (K) 5. Never use AAC for asparagine (N) 6. Always use ACG for threonine (T). Petition 870250096842, dated 10 / 23 / 2025, pp. 110 / 180 103 / 121

[0235] In this example, when the six rules for codon usage were used, they were applied in each instance where the codons D, E, G, K, N, and T appear in the encoding sequence. This approach eliminated all DRACH motifs and altered many codons that would not otherwise have been implicated in the m6A modifications.

[0236] The open reading frame (ORF) of the ZsGreen DNA sequence after applying one or more codon optimization strategies to eliminate DRACH motifs is shown below: 1. ORF ZsG following codon optimization with the best codon usage scheme (SEQ ID NO: 1) >ZsG_AllBest ATGGCCCAGAGCAAGCACGGCCTGACCAAGGAGATGACCA TGAAGTACCGGATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGATCA CCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAAC CTGTGCGTGGTGGAGGGCGGCCCCCTGCCCTTCGCCGAGGACATCCT GAGCGCCGCCTTCATGTACGGCAACCGGGTGTTCACCGAGTACCCCCA GGACATCGTGGACTTCAAGAACAGCTGCCCCGCCGGCTACACCTG GGACCGGAGCTTCCTGTTCGAGGACGGCGCCGTGTGCATCTGCAACGC CGACATCACCGTGAGCGTGGAGGAGAACTGCATGTACCACGAGAGCAA GTTCTACGGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAGAT GACCGACAACTGGGAGCCCAGCTGCGAGAAGATCATCCCCGTGCCCAA GCAGGGCATCCTGAAGGGCGACGTGAGCATGTACCTGCTGCTGAAGGA CGGCGGCCGGCTGCGGTGCCAGTTCGACACCGTGTACAAGGCCAAGA GCGTGCCCCGGAAGATGCCCGACTGGCACTTCATCCAGCACAAGCTGA CCCGGGAGGACCGGAGCGACGCCAAGAACCAGAAGTGGCACCTGACC GAGCACGCCATCGCCAGCGGCAGCGCCCTGCCCTGA; 2. ORF ZsG following codon optimization with the best codon usage scheme and then the six codon usage rules (SEQ ID NO: 2) Petition 870250096842, dated 10 / 23 / 2025, pp. 111 / 180 104 / 121 >ZsG_ABpRules ATGGCCCAGAGCAAGCACGGCCTGACGAAGGAGATGACGATG AAGTACCGGATGGAGGGCTGCGTGGATGGCCACAAGTTCGTGATCACGGGC GAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAATCTGTGCGTG GTGGAGGGCGGCCCCCTGCCCTTCGCCGAGGATATCCTGAGCGCCGCCTTC ATGTACGGCAATCGGGTGTTCACGGAGTACCCCCAGGATATCGTGGATTACT TCAAGAATAGCTGCCCGCCGGCTACACGTGGGATCGGAGCTTCCTGTTCGA GGATGGCGCCGTGTGCATCTGCAATGCCGATATCACGGTGAGCGTGGAGGA GAATTGCATGTACCACGAGAGCAAGTTCTACGGCGTGAATTTCCCCGCCGAT GGCCCCGTGATGAAGAAGATGACGGATAATTGGGAGCCCAGCTGCGAGAAG ATCATCCCCGTGCCCAAGCAGGGCATCCTGAAGGGCGATGTGAGCATGTACC TGCTGCTGAAGGATGGCGGCCGGCTGCGGTGCCAGTTCGATACGGTGTACA AGGCCAAGAGCGTGCCCCGGAAGATGCCCGATTGGCACTTCATCCAGCACA AGCTGACGCGGGAGGATCGGAGCGATGCCAAGAATCAGAAGTGGCACCTGA CGGAGCACGCCATCGCCAGCGGCAGCGCCCTGCCCTGA; 3. ORF ZsG following codon optimization with proportional usage codon usage scheme (SEQ ID NO: 3) >ZsG_AllProb ATGGCCCAGTCAAAAACATGGACTCACTAAAGAGATGACCATGAA ATACAGAATGGAGGGGTGCGTCGATGGTCACAAATTCGTCATCACCGGCGAG GGGATTGGATATCCATTCAAGGGAAAGCAGGCCATTAACCTGTGCGTCGTGG AAGGAGGACCACATGGACTCACTCATTCAGTCCATGTT GGAAACAGGGTCTTCCACTGAGTACCCACAGGATATTGTTGACTATTTTAAAAA TTCTTGCCCAGCCGGCTATACATGGGACCGCTCCTTCCTTTGAAGACGGA GCGGTGTGTATCTGCAACGCAGATATCCAGTGTCCGTTGAGGAAAACTGCA TGTATCACGAAAGCAAGTTGATTGGCCGGCCGGCCTT TATGAAGAAGATGACAGACAATTGGGAGCCCAGTTGCGAGAAAATCATTCCA GTGCCCAAGCAGGGGATTCTCAAGGGCGACGTAAGCATGTACCTGCTGCTAA Petition 870250096842, of 23 / 10 / 2025, p. 112 / 180 105 / 121 AGGATGGAGGTCGCTTACGCTGCCAGTTTGACCGTCTATAAAGCCAAGAG CGTCCCCCGGAAGATGCCGGACTGGCATTTTATCCAAGCAAGCTGACCCGT GAGGACAGGTCTGATGCCAAAAATCAGAAGTGGCACTTAACTGAGCATGCTA TCGCAAGCGGATCAGCACTCCCCGTGA; 4. ORF ZsG following codon optimization with the proportional use codon usage scheme and, subsequently, the six codon usage rules (SEQ ID NO: 4) >ZsG_APpRules ATGGCCCAGTCAAAGCATGGCCTCACGAAGGAGATGACGATGA AGTACAGAATGGAGGGTGCGTCGATGGTCACAAGTTCGTCATCACGGGCG AGGGGATTGGGTATCCATTCAAGGGCAAGCAGGCCATTAATCTGTGCGTCGT GGAGGCGGCCCACTGCCTTTCGCAGAGGATATCCTCAGTGCCGCATTCATG TATGGCAATAGGGTCTTCACGGAGTACCCACAGGATATTGTTGATTATTTTAA GAATTCTTGCCCAGCCGGCTATACGTGGGATCGCTCCTTCCTCTTTGAGGAT GGGGCGGTGTGTATCTGCAATGCAGATATCACGGTGTCCGTTGAGGAGAATT GCATGTATCACGAGAGCAAGTTCTATGGCGTGAATTTCCCTGCGGATGGGCC CGTTATGAAGAAGATGACGGATAATTGGGAGCCCAGTTGCGAGAAGATCATT CCAGTGCCCAAGCAGGGGATTCTCAAGGGCGATGTAAGCATGTACCTGCTGC TAAAGGATGGCGGTCGCTTACGCTGCCAGTTTGATACGGTCTATAAGGCCAA GAGCGTCCCCCGGAAGATGCCGGATTGGCATTTTATCCAGCACAAGCTGACG CGTGAGGATAGGTCTGATGCCAAGAATCAGAAGTGGCACTTAACGGAGCATG CTATCGCAAGCGGTTCAGCACTCCCGTGA.

[0237] Following codon optimization strategies, the number of DRACH motifs in the open reading frame of ZsGreen was quantified, the results of which are shown in Table 3 below. Table 3: Number of DRACH motifs after codon optimization of the ZsGreen ORF Codon Optimization Strategy SEQ ID NO: Number of reasons DRACH All-best 1 14 Petition 870250096842, dated 10 / 23 / 2025, page 113 / 180 106 / 121 Codon Optimization Strategy SEQ ID NO: Number of reasons DRACH All-best + 6 codon usage rules 2 0 Proportional usage 3 16 Proportional usage + 6 codon usage rules 4 0

[0238] After quantifying the number of DRACH motifs, the resulting ORFs were aligned and compared to assess the differences between the codon optimization strategies. The comparisons are shown below, where identical nucleotides are indicated with an asterisk (*), and differences in the polynucleotide sequence are shown in bold and underlined: 1. Alignment of the ZsGreen ORF following the best codon usage scheme without (SEQ ID NO: 1) and with (SEQ ID NO: 2) application of the six codon usage rules: asG-AllBast. Z$G_ABpRúléS ATGGCCGAGC.ViGCACGGCCTGACCAAÍjGAGArGACCATGAAGTACOGtrATGGAGGGC cC. ATGÜCCCAGAGCAAGCACGGCCTGACaAAGGAGATÜACGATGAAGTACCGGATGQAGGGC6Q 2sG_AlLDtsst ZsG_ABpRules TGCGTGGACGGCCACAAGirCGTGATCACCGGCGÃGRGCATCGGCrACCCCrTCAAGGGC 12 C TGCGTGGATGGCCACAAGTTCGTGATCACGGGCGAGGGCATCGGC”ACCCCTTCAAGGGC 12 □ 7,sG_AJ. IBest AAGCAGGGCATCAACCTGTGCGTGGTGGAGGGOGGCCOCCTGCCCTTCGCCGAGGAGATOm 3sG,..ABpKules AACCAGGCCArCAATCTGTGCCTCCTGGAGGGCGCCCCCCTGCCCTTCGCCGAGGATATC ISO isG^Àiia^At Zfttt_ARpPules GIGMJCâCCGCCl 'TCAiGTACGGCAACCGGGTtiTtCACCCAGTACCCCCAGGACATWiTG 24 0 CTGAGCCGGCClTInstallGTACGGCAATCGGGTGTTOACCKWJTZiCCCXX^GGATATCGTGS^C ZsG_AliBesr ZsG-ABpSules ZsG_A113eSC ZSG_AEpRUleS GACTACrTCAAGAkCAGCTGCCüCGCCGGCTACACCTGGGACCGÜAGGrrCCTGTTCGAGSCí: GATIãCriCAAG.t?.,r?.GCTGCC3CGCC3GCrA.:ACGT'3GGATCGGAGCiTCCT3TTOGÀ3 3':0 (^ACGGCGCCGTGrüCATClGCAACGCCGACATCACCGTGAGCGTGGAAGAGAACTGCATG36G GATGGCGCCGTGTGCATCI'GCAATGCCGATATCACGGTGAGCGTGGAGGAGAATTGCAIG 350 Ζ3ί;._Α11Ββ5Γ. TaCCACGAGAGC.AAGTTCrACGGCGTGAACTTCCCCGCCGACGGCCCCCTGriTGAAGAT·.'.'42C 3sG._ABpRules TACCACGAGAGCAAG1TC! ACGGCGTGAATrTCCCCGCCCÍATGGCCCCGTGA TGAAGAAG 420 ZsG-Al IBnst ATGACCGACAACTGGGAGCCCAGCTGCGAGAAGATCATCCCCGTGCCCAAGCAGGGCATC 4S0 ZsG„AHpRtiles ATGACGGATAATTGÜGAGCCCAGCTÜCtíAGAAGATCATCCCCGTGCCCAAGCAGCiGCATC 460 ZsG_AllBest CTGÍ^AGGGCGACGTGAGCATGTACCTGCTGCTGAAGGACGGCGGCCGGCTGCGGTGCCAG 54 0 sG_A0pR'i les CTGAAGGGCGATG TGAGCArGTACITTGCTGCTGÀACíGATGGCGGCCOÜGCTAGGGCGCTAG540 3sG_AllBest TTCGACACCGTGTACAAGGCCAAGAGCGTGCCCCG-3AAGATGCCCGACTGGCACTTCATC 6ü 0 ZsG_ARpRulcs TTCGATACGGTGTACAAG^CMGAGCGTGCCCCGGAftGATGCCCGATTGGCACTTCATC 6« 0 ZsG,„AJL lÊe3t ZsG..ABpRu.l es CAGC.\CAAOCTGACC€GGGAGGACCGGftaCQACGCCAAGAACCAGAAGTGGCACCTOACC650 CAGCAC.AAGCTGACGCGGGAGGATCGGAGO'JATGCCAAGAATCAGAAGTGG'.'ACCTGACG É6Ü ZsG._Al LSot GAGCACGCCATCGCCAGCGGCAGCGCCGTGCCCTGA oil ZsG_ABpRules GAGCACGCCArCGCCAÜCGGCJiGCGCCCTGCCCTGA 67 6 Á- Jt > tfc λ -Λ.· 'Ã -it i,· Λ.· JÍ- -λ Λ<C * -Ã- ·λ A ★ Λ- 'Λ -Λ -fc A· À· ’Ji· > .***· ·Λ· -Mi Petition 870250096842, dated 10 / 23 / 2025, pp. 114 / 180 107 / 121 2. Alignment of the ZsGreen ORF following the proportional codon usage scheme without (SEQ ID NO: 3) and with (SEQ ID NO: 4) application of the six codon usage rules: ZsG_AllFrob ATGGCCCAGTCAAAAACATGGACTCACTAAAGAGATGACCATGAAATACAGAATGGAGGGGG 60 Z s G_APpR tiles ATGGCCCAGTCAAAGCATGGÇCTCGAAGGAGATGACGATGAAGTACAGAATGGAGGGG60 ****** A·*** + **** A · + * A + * A * A * * κ * * * ** t *r ΐ ?< z it-λ + tw + λ ZsG_AilProb TGCGTCGATGGTCACAAAATTCGTCATCACCGGCGAGGGGATTGGATATCCATCAAGGGA120 ZsG_APpRules TGCGTCGATGGTCACAAGTTCGTCATCÃCGGGCGAGGGGATTGGGTATCCATTCAAGGGC 120 i; kk -k -f, -k -f; k íe -kk ~kk: k -k ~k -i: ): t -l·. i >r / rí Ί, t X Ir kk kkk -.* Λ Jr *rk A * * Jr **+χΙ:1;·1+·>:»:Α·ί1·Ι; ZsG_AHProb AAGCAGGCCATTAACCTGTGCGTCGTGGAAGGAGGACCACTGCCTTTCGCAGAAGATATC 180 zs G_. APpRu 1 es AAGCAGGCCATTAATClOTGCCSrCGTGGAGGGCGGCCCACTCKíCTTTÇíKíAGA&GATATC 180 * <: AA Ίτ ΐ Λ * ΐ í tk í: í * 'ti * Jí λ'í ír * * J: A· * 1 * í À * À * i * * A * i w AAA ·*· Ά •k Jr ZsG„AliProb CTCAGTüCCG^TTQXrGTATGGAAACAGGGTCTTCACTGAGTACCCAÍlAGGATATTGTT 24C Z 5 G_.APpRu 1S s CTCAGTGCCGCATTCA'FGTAT^CAATAGGGTCTTCTACCATA4GGTTGTTGTT^ JrJc+cirJr+rJrJrírJcJrJt^JrJr*·.*:**:·**;*·. Λ *.· *: SittiltltlkiA* ·* *: Jr Jr J: J; *; Jr *: Jr Λ * Jr + Λ Λ Jr Jr Λ ·* Jr ZsG_AllProb GACI?.TTTT.AAAAx\TTCTTGCCCAGCCGGCTATACATGGGACCGCTCCTTCCTCrTTGAA30C ZsG_MpRules GATTATTTTAAGAATTCTTGCCCAGCCGGCTATACGTGGGATCGCTCCTTCCTCrTTGAeSOO ZsG_AllProb gacggagcggtgtgtatctgcaacgcagatatcacagtccgttgagíjAaaactgcatg 360 ZsG_AJ?pRules GAt.GG£GCGGTGTGTATCTGCAATGCAGATATCACGGTGTCCGTTGAGGA2«ATTGCATG360 k í -it· ·*· * J< # * Ά ΐ Jr -J{ ·κ ΐ ·£ ír λ * * AA· w * ·% ·& & « +. 4c Jr A * AAA Jr ·» +. AA Λ ·* * AA * * AA h * * A ZsG._A.llProb TATC^CGAAAGCAAGTTCTATGGCGTGAATTICCCrGCGGACGGGCCCGTTATGAAGAAG42G Z 3 G„APpRu1e s TATCACGAGAGCAAGTTCTATGGCGTGAAiiTCCCrGCGGATGGGCCCGTTATGAAGAAG^ZC ZsG_Allxob ATGACJiGACAATTGGGAGCCCAGTTGCGAGAAAATCATTCCAGTGCCCAAGCAGGGGATI 480 ZsG_A£*pRules ATGACGGATAATTGGGAGCCCAGTTGCGAGAAGA'rCATTCCAGTGCCCAAGCAGGGGATr480 » + + ** -r +&'r &&&&*: Ir-kk&k-kkkk-ir-kk-it -kk-ti'k-kKk-kkii'k-kir-'r-k'is-kk-kk-k'k-kkkkx ZsG_AllFrob CTCAAGGGCGACGTAAGCATGTACCTGCTGCTAAAGGArG'OAGGTCGCTTACGCTGCCAG 5 G 4 G 3 e CTCAAGGGCGATGTAAGCATGTACCTGCTGCIAAAGGATGGCGGTCGCTTACGCTGCCAG540 ZsG_.AllProb TTTGACACCGTCTATAAAGGCAAGAGCGTCCCCCGGAAGATGCCGGACTGGC\ATrTTATC 600 Z sG„ APpRu.l es TTTGATACGGTCTATAAGGCCAAGAGCGTCCCCCGGAAGATGCCGGATTGGCATTTTATC600 ***** ** ******** ***************************** * * ** +, * * * *·* * * ZsG-AUProb CAGCACAAGCTGACCCGTGAGfâACAGGTCTGATCAAGAAGTAGTAGTAGTAG600 s600 G„ AP pRu 1 is CAGCACAAGCTGACecGTGa.GGAT6.GGTCTGATGCCAAgA?»rCAGaAGTGGCÃCTTIAC6 660 * ****·***.'**'*'**'* **'*****''* ***'*·*·*'******** ****·****·**** ******** ZsG_AllProb GAGCATGCTATCGCAAGCGGATCAGCACTCCCGTGA 696 ZsG_APpRules GAGCATGCTATCGCAAGCGGTTCAGCACTCCCGTGA 698 ******************** ***************.

[0239] Each of the four ORFs of the ZsGreen fluorescent protein Petition 870250096842, dated 10 / 23 / 2025, pp. 115 / 180 108 / 121 was then commercially ordered as synthetic double-stranded DNA (dsDNA) fragments and cloned into the pAAV ZsGreen1 plasmid between the BamHI and EcoRI restriction sites (FIG. 1). After verifying the sequences of the cloned plasmids by Sanger sequencing, the ZsGreen1 plasmid variants, along with a pUC19 control plasmid to equalize the total DNA amounts, were transiently transfected into HEK293T cells in 24-well plates with lipofectamine 3000, according to the manufacturer's instructions. The DNA transfection conditions are shown in Table 4 below. Table 4: Transfection conditions Transfection dilution condition pUC19 DNA per well (μ9) ZsGreen variant DNA per well (μ9) 1:4 0.4 0.1 1:16 0.47 0.029

[0240] Transfected HEK293 cells were collected by trypsinization 2 or 3 days after transfection, and the relative protein production for each ORF variant under both transfection conditions was analyzed by flow cytometry, in which the relative fluorescence of ZsGreen in the green channel was measured. ORFs generated from the best codon usage scheme with or without implementation of the six additional codon usage rules were compared after transfection (FIG 2). ORFs generated from the proportional usage scheme with or without implementation of the six additional codon usage rules were also compared (FIG 3). Example 2. Determination of GLP-1-Fc protein yield using a commercially available web-based codon optimization tool with or without reducing DRACH motifs.

[0241] This example describes the codon optimization process of a sequence encoding a GLP-1-Fc protein after codon optimization with a commercially available web tool and comparatively analyzing the resulting sequences and protein production with or Petition 870250096842, dated 10 / 23 / 2025, pp. 116 / 180 109 / 121 without applying the codon usage rules to reduce or eliminate DRACH motifs. The codon usage rules, or pRules, are as follows: 1. Never use GAC for aspartic acid (D) 2. Never use GAA for glutamic acid (E) 3. Never use GGA for glycine (G) 4. Never use AAA for lysine (K) 5. Never use AAC for asparagine (N) 6. Always use ACT for threonine (T).

[0242] In this example, the six codon usage rules were applied only to observed DRACH motifs and not to all instances where the codons D, E, G, K, N, and T appear in the sequence. To generate a codon-optimized GLP1-Fc ORF for protein production, the amino acid sequence was entered into the IDT Codon Optimization Webtool available on the Integrated DNA Technologies website. After entering the amino acid sequence, the GLP-1-Fc ORF was generated and the DRACH motifs were quantified. The GLP-1-Fc ORF generated by the IDT Codon Optimization Webtool (SEQ ID NO: 5) is shown below: IDT ATGATCCCCGCAAAGGACATGGCGAAGGTAATGATTGTGATGC TGGCAATCTGCTTTCTGACTAAGTCAGACGGGAAGTCAGTTAAGAAGCGCCA CGGCGAAGGCACCTTTACCAGTGATGTTAGCTCGTATCTGGAGGAACAGGCC GCCAAAGAGTTCATCGCCTGGCTCGTGAAGGGGGGAGGCGGAGGAGGTGG ATCTGGGGGCGGTGGGTCCGGTGGTGGCGGCTCCGCGGAATCGAAATACG GCCCTCCTTGTCCACCATGCCCCGCTCCTGAAGCCGCAGGAGGACCCTCTGT CTTTCTCTTCCCACCTAAGCCTAAAGATACTCTTATGATAAGTCGAACCCCTGA GGTGACATGCGTGGTGGTCGATGTTTCACAGGAAGACCCTGAGGTCCAGTTC AACTGGTATGTTGATGGGGTAGAGGTACACAATGCCAAAACAAAGCCTAGAG AGGAACAGTTCAATAGCACATATCGCGTGGTTTCCGTGCTGACAGTGCTGCA Petição 870250096842, de 23 / 10 / 2025, pág. 117 / 180 110 / 121 CCAGGACTGGCTCAACGGAAAGGAGTACAAGTGTAAGGTTAGCAACAAAGGC CTTCCAAGTTCGATAGAGAAGACAATCTCTAAAGCTAAGGGACAGCCTAGAGA GCCACAGGTTTACACTCTGCCCCCCAGTCAGGAGGAAATGACCAAAAACCAG GTCAGCCTGACTTGTCTGGTGAAGGGATTCTATCCATCAGATATTGCCGTCGA GTGGGAAAGCAATGGGCAGCCTGAGAATAATTACAAAACCACACCCCCCGTG CTGGATAGCGACGGCAGCTTTTTCCTATATTCCCGACTTACCGTTGACAAGTC CCGCTGGCAGGAAGGCAATGTGTTTTCTTGCAGTGTGATGCACGAGGCATTG CATAATCACTATACCCAGAAAAGCCTGAGCCTGTCCCTGGGGTGA.

[0243] A total of 19 DRACH motifs appeared in the GLP1-Fc ORF. The ORF was then modified according to the six rules for codon usage to modify the DRACH motifs. Following these rules, 18 DRACH motifs were removed by synonymous codon substitution. Due to cloning limitations, one DRACH motif in the sequence could not be altered and was left in the ORF without substitutions. The resulting modified sequence in which all but one DRACH motifs were removed from the IDT sequence (IDT-m6A) (SEQ ID NO: 6) is shown below: IDT-m6A ATGATCCCCGCAAAGGATATGGCGAAGGTAATGATTGTGATGCT GGCAATCTGCTTTCTGACGAAGTCAGACGGGAAGTCAGTTAAGAAGCGCCAC GGCGAAGGCACCTTTACCAGTGATGTTAGCTCGTATCTGGAGGAGCAGGCCG CCAAAGAGTTCATCGCCTGGCTCGTGAAGGGGGGAGGCGGAGGAGGTGGAT CTGGGGGCGGTGGGTCCGGTGGTGGCGGCTCCGCGGAATCGAAATACGGC CCTCCTTGTCCACCATGCCCCGCTCCTGAAGCCGCAGGAGGCCCCTCTGTCT TTCTCTTCCCACCTAAGCCTAAAGATACTCTTATGATAAGTCGAACGCCTGAG GTGACGTGCGTGGTGGTCGATGTTTCACAGGAGGATCCTGAGGTCCAGTTCA ACTGGTATGTTGATGGGGTAGAGGTACACAATGCCAAGACGAAGCCTAGAGA GGAGCAGTTCAATAGCACATATCGCGTGGTTTCCGTGCTGACGGTGCTGCAC CAGGATTGGCTCAACGGAAAGGAGTACAAGTGTAAGGTTAGCAACAAAGGCC Petição 870250096842, de 23 / 10 / 2025, pág. 118 / 180 111 / 121 TTCCAAGTTCGATAGAGAAGACGATCTCTAAAGCTAAGGGCCAGCCTAGAGA GCCACAGGTTTACACTCTGCCCCCCAGTCAGGAGGAAATGACGAAGAATCAG GTCAGCCTGACGTGTCTGGTGAAGGGATTCTATCCATCAGATATTGCCGTCG AGTGGGAAAGCAATGGGCAGCCTGAGAATAATTACAAGACGACACCCCCCGT GCTGGATAGCGACGGCAGCTTTTTCCTATATTCCCGACTTACCGTTGATAAGT CCCGCTGGCAGGAAGGCAATGTGTTTTCTTGCAGTGTGATGCACGAGGCATT GCATAATCACTATACCCAGAAAAGCCTGAGCCTGTCCCTGGGGTGA.

[0244] The GLP1-Fc ORFs generated in which DRACH motifs were left alone (IDT) (SEQ ID NO: 5) or removed after application of the six codon usage rules (IDT-m6A) (SEQ ID NO: 6) were compared and are shown below, in which identical nucleotides are indicated with an asterisk (*) and differences in the polynucleotide sequence are shown in bold and underlined: IDT I'TXGGCAJiAGAATTGGGATTCGCGAGAATTCGCCACCATGATCCCCGCAAAGGACATGGC 60 IDT-môA rTIGGCAAAGAATTGGGATTCGCGA.GAATTCGCCACCATGATCCCCGCAAAGGATArGGC 60 IDT GAAGGTAATGATTGTGATGCTGGCAÂTCTGCTTTCIGACTAAGTCAGACGGGAAGTÇAGT 120 IDT-ITiôA GAAGGTAAl'GATTGTGATGCTGGCAArCTGCTTTC'l'GACGAAGTCAGACGGGAAGTCAGT 120 idt TAAGAAGCGCCACGGCGAAGGCACCTTTACCAGTGATGTTAGCTCGrATCTGGAGGftACA 180 IE>T--m6A TAAGAAGCGCCACGGCGAAGGCACCTTTACCAGTGATGTTAGCTCGTATCTGGAGGAGCA 180 + ΐ Λ À A it ΐ t AAA i ΛΑ AAA íri ίΆ Λ k AA + ΑΛ Λ A *k Α Λ At Α< Λ* ~hk At Ar + λ* *Â 4: At Ac k IDT GGCCGCCAAAGAGTTCATCGCCTGGCTCGTGAAGGCSGGGAGGCGGAGGTGGATCTGG 240 TDT-móA GGCCGCCAAAGAGTTCATCGCCTGGCTCGTGAAGGGGGGAGGCGGAGGAGGrGGATCTGG •fc k -k * kk Ar Ar Ar kkk -k W k Ar -k * Ar k Ar k Ac 4: k + k Αν 4: » Ac kkk * Ar kk -fc k At Ar k Ar t>r At Sr kkk * Ar + ».· Year 4: Year ·<· Ac k IDT GGGCGGTGGGTCCGGTGGTGGCGGCTCCGCGGAATCGAAATACGGCCCrCCTTGTCCACC 309 IDT-m6A GGGCGGTGGGTCCGGTGGTGGCGGCTCCGCGGAATCGAAATACGGCCCTCCTTGTCCACC 30Ü IDT ATGCCCCGCTCCTGRAGCC^AGGAGGACCCTCTGTCTTTCTCTTCCCACTAãGCCTAA IDT-mSA ATGCCCCGCrCCTGÂA.GCCGCAGGÀG(^CCCTCTGTCXTTCTGTTCCC.aCCTÃAGCCCTÂA 3 60 * *» *** * ** *** lt r* * * * * t * ** # * A · * * * * » kk * k Ar·*:* ** ·k A Ar k- * k * * k * + At Ar AGATAClCTT.ATGATAaGTCGAACCCCTGAGGTGACAXGCC7TGGTGGTCGATGTTTCACA 420 IDT-mSA AfíATACrcrrATGATAAGTCGAACGCCTGAGGTGACGTGCGTfíGTG<5TCGATGTTTCACA 420 * tk r AtAr k I#** ♦ it wit ♦ k *» *t 4 At k * At Ar k *-k -k Ac -k sr V + k Ar K -k -k Λ · Λ · Ar. Ar Ar A> -Λ- 4 k· ie k * Ac Ar Ac IDT GGAAGACCCTGAGGTCCAGTTCAACTGGTATGTrGATGGGGTAGAGGTACACAATGCCAA 430 IDT-tfi6A GGAGGATCCTGAGGTCCAGTTCAACTGGTATGTTGATGGGGTAGAGGTACACAATGCCAA 480 Petition 870250096842, of 23 / 10 / 2025, p. 119 / 180 112 / 121 IDT AACAAAGCCIAGAGAGGAACAGrTCAATAGCACATATCGCGrGGrXTCCGTSCTGACAGT 54C IDT-rrtóA GACGAAGCCTAGAGAGGAGCAGTTCA / irAGCACATriTCGCGTGGTTTCCGIGCXGACSGr 540 IDT GCTGCACCAGGACTGGCTCAACGGAAAGGAGTACAAGTGTAAGGTTAGCAACAAAGGCCT600 IDT-m6A GCTGCACCAGGATTGGCICAACGGAAAGGAGTACAAGTGTAAGGTTAGCAACAAAGGCCT600 -ft*#*******·** »***ΐ**^·Μ*·*ΐ*Ο**·#ί**+***Μίί***·***:*Ο·ί****·*ί IDT ITACACTCTGaCCCCCAGTTCAGGAGGAAATGACCAAAÃACAGi^TCAGCCCTGACTTGrcr 72C XDT-mSA ITACACrCTGCCCGCCAGTCAÍsGAGGÂAATGACGAACÃATCAGGTCAGCCTGACGTCÍTCr 720 IDT GGTGGAGGGÀTTCTATCCATCAGATATTGCCGTCCAGTÜGGAAAGCAATGGGCAGCCrGA730 II?T-iri6A GGIGAAGGGATTClATCCATCAGATA.TTGCCGrCGÀGTGGGAAAGCAATGGGCAGCCrGA78C TDT GAATAATTACAAAACCACACCCCCCGTGCTGGATAGCGACGGCAGCTTTTCCTATArTC 340 IDT--m&A GAATAATTACAAGACGACACCCCGTGCTGGATAGCGACGGCAGCTTTTICTATATArTC 340 í***lr*****H w* *******)(>******************··***************·** TDT CCGACTTACCGTTGACAAGTCCCGCTGGCAGGAAGGCAATGTGTTTTCTTGCAGTGTGAT 900 IDT--m»A CCGfiCTTACCGTTGATAAGrCCCGCTGGCAGGAÃGGCAÃIGTGTTTTCTTGCAGTGTGAT 900 IDT ÜCACGAGGCATTGCATAATCACTATACCCAGAAAAGCCTGAGCCTGTCCCTGGGGTGATA 960 TDT-mSA GCACGAGGCATTGCATAATCACT?i.TACCCAGAAAAGCCTGAGCCTGTCCCIGGGGTGATA 96C IDT ÀGGAXCCACGGGTGGCATCCCXGTGACCCCTC 992 IDT~m5A AGGATCCACGGGGTGGCATCCCTGTGACCCCTC 992

[0245] The two ORFs shown above were ordered as synthetic dsDNA fragments and cloned into the pAAV ZsGreenl plasmid between the BamHI and EcoRI restriction sites (FIG. 1). After verifying the sequences of the cloned plasmids by Sanger sequencing, the plasmids containing the ORF variants, along with a control pUC19 plasmid for a total amount of 0.5 pg of DNA, were transiently transfected into HEK293T cells in 24-well plates with lipofectamine 3000, according to the manufacturer's instructions. The DNA transfection conditions are shown in Table 5 below. Table 5: Transfection Conditions pUC 19 transfection dilution condition DNA per well (pg) ORF DNA variant GLP-1-Fc per well (pg) 1:10 0.45 0.05 Petition 870250096842, dated 10 / 23 / 2025, pp. 120 / 180 113 / 121

[0246] Two days after transfection, cell culture supernatants were collected and the amount of secreted GLP-1-Fc protein was quantified via ELISA. The concentration of GLP-1-Fc protein detected increased more than 5 times in the sample in which the DRACH motifs were reduced, compared to the sample whose codon was optimized only with the use of the web IDT tool (FIG. 4). Example 3. Determination of the yield of GLP-1Fc protein production via AAV transduction after elimination of DRACH motifs.

[0247] This example describes the codon optimization process of a sequence encoding a GLP-1-Fc protein after codon optimization, in which all amino acids in the ORF were coded by the most preferred codon (i.e., the highest frequency of use) in each instance. The codon-optimized ORF was then subjected to codon usage rules to eliminate DRACH motifs. The codon usage rules, or pRules, are as follows: 1. Never use GAC for aspartic acid (D) 2. Never use GAA for glutamic acid (E) 3. Never use GGA for glycine (G) 4. Never use AAA for lysine (K) 5. Never use AAC for asparagine (N) 6. Always use ACG for threonine (T).

[0248] The codon-optimized ORF encoding GLP-1-Fc (denoted CH) (SEQ ID NO: 7) contains 19 DRACH motifs and is shown below: CH Atgatccccgccaaggacatggccaaggtgatgatcgtgatgctggccatctgcttcctgacca agtccgacggcaagtccgtgaagaagcgccacggcgagggcaccttcaccagcgacgtgtctagctacctggag gagcaggccgccaaggagttcatcgcctggctggtgaagggcggcggcggcggcggcggcagcggcggcggc ggcagcggcggcggcggcagcgccgagagcaagtacggccccccctgccccccctgccccgcccccgaggcc gccggcggccccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtg Petition 870250096842, dated 10 / 23 / 2025, pp. 121 / 180 114 / 121 acctgcgtggtggtggacgtgagccaggaggaccccgaggtgcagttcaactggtacgtggacggcgtggaggtg cacaacgccaagaccaagcccagggaggagcagttcaacagcacctacagggtggtgagcgtgctgaccgtgc tgcaccaggactggctgaacggcaaggagtacaagtgcaaggtttctaacaagggcctgcccagcagcatcgag aagaccatcagcaaggccaagggccagcccagggagccccaggtgtacaccctgccccccagccaggagga gatgaccaagaaccaagttagcctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagtggga gagcaacggccagcccgagaacaactacaagaccaccccccccgtgctggacagcgacggcagcttcttcctgt acagccgactgaccgtggacaagagcaggtggcaggagggcaacgtgttcagctgcagcgtgatgcacgaggc cctgcacaaccactacacccagaagagcctgagcctgagcctgggctga.

[0249] 0 CH ORF was further modified following the six rules of codon usage to eliminate DRACH motifs, resulting in 20 base pair substitutions and the removal of all 19 DRACH motifs that were present in the codon-optimized CH ORF. The additional modified ORF encoding GLP-1-Fc in which all DRACH motifs have been removed (denoted CH-m6A) (SEQ ID NO: 8) is shown below with all modified base pairs shown in bold and underlined: CH-m6A atg ate cee gcc aag gaT atg gcc aag gtg atg ate gtg atg ctg gcc ate tgc ttc CTG ACG aag tcc gac ggc aag tcc gtg aag aag cgc cac ggc gag ggc acc ttc acc age gac gtg tet age tac ctg gag gag cag gcc gcc aag gag ttc ate gcc tgg ctg gtg aag ggc ggc ggc ggc ggc ggc ggc age ggc ggc ggc ggc age ggc ggc ggc ggc age gcc gag age aag tac ggc ccc ccc tgc ccc ccc tgc ccc gcc ccc gag gcc gcc ggc ggc ccc age gtg ttc ctg ttc ccc ccc aag ccc aag gaT acc ctg atg ate age aGG ACG cee gag gTG ACG tgc gtg gtg gtg gac gtg age cag gaG GAT Ccc gag gtg cag ttc aac tgg tac gtg gac ggc gtg gag gtg cac aac gcc aAG ACG aag ccc agg gag gag eag ttc aac age acc tac agg gtg gtg age gtg cTG ACG gtg ctg cac cag gaT tgg ctg aac ggc aag gag tac aag tgc aag gtt tcT AAT Aag ggc ctg ccc age age ate gag aAG ACG ate age aag gcc aag ggc cag ccc agg gag ccc cag gtg tac acc ctg ccc ccc age cag gag gag aTG ACG aaçL aaT ca a gtt age CTG ACG tgc ctg gtg aag ggc ttc tac ccc age gac ate gcc gtg gag tgg gag age aac ggc cag ccc gaG AATAaT tac aAG ACG acc ccc ccc gtg ctg gaT age gac ggc age ttc ttc ctg tac age ega cTG ACG gtg gaT aag age agg tgg cag gag ggc aac gtg ttc age tgc age gtg atg cac gag gcc ctg cac aac cac tac acc cag aag age ctg age ctg age ctg ggc tga

[0250] The alignment of codon-optimized (CH) GLP-1-Fc ORF (SEQ ID NO: 7) and codon-optimized GLP-1-Fc ORF with removal of DRACH motif sequences (CH-m6A) (SEQ ID NO: 8) are shown below, Petition 870250096842, dated 10 / 23 / 2025, pp. 122 / 180 115 / 121 where identical nucleotides are denoted with an asterisk (*), and the sequence CH-m6A is shown with the nucleotide sequence that made up the DRACH motif in uppercase letters and the modified nucleotide in bold and underlined: CH atgetcaccgcca0ggaGatggccaaggtgat.gat.cgtgatgctggccatct.gc.tt.cctg CH-mfíA atgatccccgcca&ggaT«tggcceaggtgatgatcgtgatgctggccatctgcttccTG accaagtccgacggcaagtccgtgaagaagcgccacggagagggQaGcttcaccagcgac120 Ca-m6A ACGaagtccgacggcaagtccgtgaagaagcgccacggcgagggcaccttcaccagcgac + + * +· +· k + ft + 'κ + + + + i< + 'k ·>: + 'k -Jr + + + + * ·*: +· + + + + + * + A* + + * + * gt. acctggaggagaggccgccaaggagtcatcgcctggctggtgaagggc180 CH-ra6A gtgtctagctacctggaggagcaggccgcoaaggagttcgectggctggtgaagggc180 •t ii * A w rfkr * 4 k J* Ί + ΧΧΛ * 4. / , 1· * χ ΐ Ί + Ί- yxü + 1 + i · · · ϛ ggcggcggcggcggcggcggcggcggcggcggcggcggcgcgcgc240 CH-mtA ggcggcggaggcggcggcggcggcggcggcggcggc 240 + + + +++- + ++ + ++ ++ + + + ++ ++ + + + ++ + ++ ++ ++-+ + + + + + +-++ + + + +++++ni + +++- + aagtacggccccccctgccccccgcgg CH-mbA aagtacggccccccctgccccccctgccccgccccgaggccgcggcggcccgtg300 *+++*++++++++++**++**+++++++++++++++++++++*++++++++++++++++*+*+++++++++++++ ttcctgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacc360 CH-m€A ttcctgttcccccccaagccca agga®accctgatgatcagc«GGACGcccgaggTGACG360 **»-** + + * + + ** + + ***+*** + * + * ** + + + *** + **+*«* + + ** + *******+ + + + tgcgtggtggtggíicgtgí.gccíggaggacc-ccgíiggtgcagtt.ct.actggt: acgtgg <sc CH-ai6A tgcgr,ggtggtggaagr,gagccKggaGGATCccgaggtgcagt:tc^actggt .acgtggac420 + + x + +. + + + + + + * i- A + + + * + * + + + A· + + + * + + * + *- * + + *· + + + + * A- + + * + -A *- * + + A· ·« + + ggcgtggaggtgcaoaacgceaagaceaagcceagggaggagcagttcaacagoaccuac:480 C3-ai6A ggcguggaggugcaaaacgccaAGACGaagacc&gggaggagcagttcaacagcacctac480 CH agggtggtgagcgtgctgaccqtgcaccaggactggctgaacggcaaggagtacaag540 CH~ai6A agggtgqtigagcgtgcTGACGqt get gcaccaggaTtggctgaacggcaaggagt acaag540 t.gcaaggtttctaacaagggcctgcccagcagcatcgagaagaccstcagcaaggccag600 c?i--n6A tgcaaggtttcTAa.TAagggcctgcccagcagca-t egaga AGACgatcagcagcagcagcag600 +++++++++++++++++++++++++ ++++++++++++++++++++++++++++-+ +++++++++++++++ ggccagcccagggagccccaggtgtacaccatgcoccccagccagga*gga.gatgaccaag CH->6A ggccagccccaggggagcccoeggtgtacaccctgccccccagccaggaggaTGACGaag + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Ch aaocaagttagcctgagctgcctggtgaagggcttictaccccagcgacatogccgtggag 720 CH“in€A eaTcaiagttagcc'rGACG'-gcctgqtgaaqggcttctaccccagccacatcqccgtggeg 7 20 + + + + + + + + + + + * + + ++ *·+ + + + +* + + + + + + + + *·+ + + + + + + + + + + + + + + + * + * + * + * tgggagagcaacgecagcccgaacaactacacccccccgtgctggacagc CH -mt A tggge.gagcaii cggccaqeccgaGAATAaTt.acaAGACGÁacc{?cccccgtgct ggaTagc. + + CH-ΓΰΰΑ gacggcagcttct-t cctgtac«gccgacTGACGgtgg«Taa.gagcaggtggcaggagggc 340 Petition: 870250096842, on 10 / 23 / 2025, p. 123 / 180 116 / 121 aacgt.gttcagctgcagegtgatgcacgaggc.ee gcacaaccactacacccagaagagc :H-m6A aacgtgttcagetgcagcgtgatgcacgaggccctgcacaaccactacacccagaagage 900 kk 9c k 9c xkk 9: 9: ·*.' it * St * kkk 9c k * 9c Λ kk 9? 9; kkk Λ xkk 9c Λ' * 9c Λ 9τ 9c 9c *.' k 9c 9c k Ά k it 9c Λ* 9c kkkk 9c kk CH ctgagcctgagcctgggctga 921 CH-M6A ctgagcctgagcctgggctga 921

[0251] After validating the ORF polynucleotide sequences, the amino acid sequences encoded by each ORF were aligned to ensure that the GLP-1-Fc protein maintained 100% sequence identity after the elimination of DRACH motifs in the ORF. The alignment of the amino acid sequences of the CH ORF (SEQ ID NO: 9) and the CH-m6a ORF (SEQ ID NO: 10) are shown below, where identical amino acids are indicated with an asterisk (*): CH MIPAKDMAKVMIVMLAICFLTKSDGKSVKKRHGEGTFTSDVSSYLEEQAAKEFXAWLVKG60 CH-m6A MIPAKDMAKVMIVMLAICFI.TKSDGKSVKKRHGEGTFTSDVSSYI1EEQAAKEFIAWLVKG60 CH GGGGGGSGGGGSGGGGSAÊSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT120 CH-m6A GGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT120 CWVDVSQEDPEVQFHWYVDGVEVHNAKTKPREEQFNS T YRWS VLTVLHQDWLNGKE YK180 CH-m6A CVWDVSQEPPSVQFNtJYVPGVSVHNAKTKPRESQFNSTYRVVSVLTVLHQDWLNGKEYK180 CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSI.TCI,VKGFYPSDIAVE CH-m6A CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE ·* kkkk * kkkkkkkkkk Ά kk fr A k k. k 9c kkkkkkkkkkkkkkkkkk k Ά kkkkkkkkkkk k. CH WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS300 CH-m6A WESNGQPENN¥KTTPPVI.DSDGSFFI.YSRI.TVDKSRWQEGNVFSCSVMHEAI>HNHYTQKS -A- CH LSLSLG* 306 CH-m6A LSLSLG* 306

[0252] After confirmation of the polynucleotide and amino acid sequences, the CH and CH-m6A GLP-1-Fc ORFS (SEQ ID NOs: 7 and 8) were formed Petition 870250096842, 10 / 23 / 2025, p. 124 / 1 117 / 121 were sorted as synthetic dsDNA fragments and subsequently cloned into the pAAV ZsGreen1 plasmid between the BamHI and EcoRI restriction sites. The sequences of the cloned plasmids were verified by Sanger sequencing.

[0253] The CH and CH-m6A plasmids, as well as the IDT and IDT-m6A plasmids described in Example 2, were then constructed as AAV9 vectors by triple transfection in HEK293 cells using standard methods known in the field. The recovered AAV9.GLP-1Fc vectors were titrated by qPCR to determine vector genome concentrations and then applied to HEK293 cells at a multiplicity of infection (MOI) of 1 x 10⁶ vector genomes per cell for GLP-1-Fc protein transduction. After 3 days of protein transduction, the supernatants from the HEK293 cell cultures were collected, and the levels of secreted GLP-1-Fc protein were determined by ELISA.

[0254] GLP-1-Fc protein levels in each sample were compared to the codon-optimized CH ORF sample (FIG. 5). The CH-m6A sample, in which all DRACH motifs were removed from the codon-optimized CH ORF (SEQ ID NO: 8), resulted in a 3-fold increase in detectable GLP-1-Fc protein secretion compared to the CH sample, in which the CH ORF (SEQ ID NO: 7) was used. The codon-optimized ORF using the IDT codon optimization web tool (SEQ ID NO: 5) exhibited a 1.3-fold increase in detectable GLP-1-Fc protein secretion compared to the CH sample. Furthermore, the optimized ORF of the IDT codon in which all but one DRACH motif were removed (SEQ ID NO: 6) resulted in a 6.8-fold increase in detectable secretion of GLP-1-Fc protein compared to the CH sample. Example 4. Evaluation of circulating GLP-1-Fc protein in vivo after AAV administration.

[0255] This example describes the impact that the different Petition 870250096842, dated 10 / 23 / 2025, pp. 125 / 180 118 / 121 codon optimization methodologies described in Examples 2 and 3 have applications in in vivo protein production.

[0256] In addition to the 4 AAV vectors prepared as described in Example 3, a native ORF encoding GLP-1-Fc (i.e., an ORF without the application of any codon optimization method) was generated based on the human GLP-1 gene sequence. The native GLP-1-Fc ORF (SEQ ID NO: 11) is shown below: native ATGATACCTGCAAAAGACATGGCTAAAGTTATGATTGTCATGT TGGCAATTTGTTTTCTTACAAAATCGGATGGGAAATCTGTAAAGAAGCGGC ATGGAGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGAACAAG CTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCGGAGGAGGGGGGGG GGGGTCAGGCGGAGGGGGCTCCGGAGGAGGAGGATCTGCAGAGTCCAA ATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGGCTGCAGGGGGA CCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACC CCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCC ATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGA GGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT ACACACAGAAGAGCCTCTCCCTGTCTCTGGGTTGA.

[0257] The plasmid and AAV vector encoding the native sequence Petition 870250096842, dated 10 / 23 / 2025, pp. 126 / 180 119 / 121 GLP-1-Fc were generated and validated using the same methods described in Example 3.

[0258] To test the in vivo protein production of the four described codon optimization methods, each of the described GLP-1-Fc AAV vectors was injected into a mouse at a low dose (1 x 109 genome copies (GC) per mouse) or a high dose (1 x 1010 GC per mouse). Eight 8-week-old female mice comprised each vector and experimental dosage group.

[0259] Each mouse received a single intramuscular injection of 10 μL of a GLP-1-Fc AAV vector in the leg. A serum sample was collected from the mice every 2 weeks for 60 days after the injection. Muscle tissue at the injection site, as well as a liver sample, were collected at necropsy, as these tissues were the main organs for protein transduction after intramuscular AAV administration. Serum GLP-1-Fc protein levels were assessed by ELISA, and GLP-1-Fc mRNA transcription levels in muscle and liver tissue homogenates were measured by qPCR.

[0260] In both dosage groups, the CH-m6A and CH codon-optimized AAV vectors produced the highest concentrations of detectable GLP-1-Fc protein in serum at all time points over 42 days (FIGS. 6A-6B). On day 14, serum GLP-1-Fc protein levels were lowest in samples containing the vector encoding the native sequence and highest in samples containing the vector encoding the optimized CH codon sequence in which the DRACH motifs were removed (CH-m6A) (SEQ ID NO: 8). The vector encoding the CH-m6A sequence (SEQ ID NO: 8) led to serum concentrations of GLP-1-Fc protein that were 4.5 times higher compared to the vector encoding the native sequence (FIG. 7).

[0261] To better assess the impact of m6A modifications (or Petition 870250096842, dated 10 / 23 / 2025, pp. 127 / 180 120 / 121, i.e., the presence of DRACH motifs) on protein production efficiency, detectable serum GLP-1-Fc protein levels were pairwise compared based on codon optimization strategy and dosage group 28 days after AAV vector administration (FIGS. 8A-D). Three of the four experimental groups compared showed increased serum GLP-1-Fc protein levels when DRACH motifs were removed. The IDT codon optimization scheme after DRACH motif removal (SEQ ID NO: 6) resulted in a 3.6-fold increase in serum GLP-1-Fc protein levels compared to the IDT codon optimization scheme alone (SEQ ID NO: 5) after low-dose injection of each AAV vector (FIG. 8A).The CH codon optimization scheme after DRACH motif removal (SEQ ID NO: 8) resulted in a 1.75-fold increase in serum GLP-1-Fc protein levels compared to the CH codon optimization scheme alone (SEQ ID NO: 7) after low-dose injection of each AAV vector (FIG. 8B). Similarly, at high doses, the IDT codon optimization scheme after DRACH motif removal (SEQ ID NO: 6) resulted in a 2.0-fold increase in GLP-1-Fc protein levels compared to the ID codon optimization scheme alone (SEQ ID NO: 5) (FIG. 8D). However, no significant differences in serum GLP-1-Fc protein levels were observed with high-dose injection of any variation of the optimized CH codon vectors.

[0262] A similar trend was observed for GLP-1-Fc mRNA transcription levels in muscle and liver tissue. In all groups except the low-dose CH codon-optimized vector, vectors in which DRACH motifs were removed after codon optimization produced more detectable GLP-1-Fc mRNA transcripts (FIGS. 9-10). The greatest difference in tissue-specific GLP-1-Fc mRNA transcript expression was observed in muscle tissue after high-dose injection of the IDT codon-optimized vectors, in which the DRACH motifs were removed from the ORF. Petition 870250096842, dated 10 / 23 / 2025, pp. 128 / 180 Codon-optimized 121 / 121 led to a 15.2-fold increase in detectable levels of GLP-1-Fc mRNA after 60 days (FIG. 9C). Collectively, these results indicate that removal of DRACH motifs can lead to an increase in the expression of mRNA transcripts and proteins after transduction. Other Modalities

[0263] All publications, patents and patent applications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each independent publication or patent application were specifically and individually indicated for incorporation by reference.

[0264] Although the invention has been described in connection with specific embodiments thereof, it will be understood that it is also susceptible to further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that fall within the known or customary practice of the art to which the invention belongs and that can be applied to the essential features set forth above in this document, and follows within the scope of the claims.

[0265] Other modalities are within the scope of the claims. Petition 870250096842, dated 10 / 23 / 2025, pp. 129 / 180

Claims

1 / 27 CLAIMS 1. A METHOD FOR CODON OPTIMIZATION OF A POLYNUCLEOTIDE SEQUENCE encoding a polypeptide of interest, the method characterized in that it comprises replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence with an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide.

2. METHOD, according to claim 1, characterized in that the polynucleotide sequence comprises a plurality of DRACH motifs and in which the substitution is performed only on a subset of the DRACH motifs.

3. METHOD, according to claim 2, characterized in that the substitution is performed only on a DRACH motif that is separated from a splice junction site within the polynucleotide sequence by at least 10 nucleotides.

4. METHOD, according to claim 3, characterized in that the substitution is performed only on a DRACH motif that is separated from a splice junction site within the polynucleotide sequence by at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 180 nucleotides, at least 190 nucleotides, at least 200 nucleotides or more.

5. METHOD, according to claim 2, characterized Petition 870250096842, dated 10 / 23 / 2025, page. 130 / 180 2 / 27 because the substitution is performed only on a DRACH motif that is separated from a splice junction site within the polynucleotide sequence by about 10 nucleotides to about 200 nucleotides, by about 20 nucleotides to about 190 nucleotides, by about 30 nucleotides to about 180 nucleotides, by about 40 nucleotides to about 170 nucleotides, by about 50 nucleotides to about 160 nucleotides, by about 60 nucleotides to about 150 nucleotides, by about 70 nucleotides to about 140 nucleotides, by about 80 nucleotides to about 130 nucleotides, by about 90 nucleotides to about 120 nucleotides or by about 100 nucleotides.

6. METHOD, according to any one of claims 1 to 5, characterized in that the substitution is carried out on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, TGACT, AAACA, AAACC, AAACT, AGACC, GAACA, GGACC, TAACA, TAACC, TAACT, TGACA and TGACC.

7. METHOD, according to claim 6, characterized in that the substitution is carried out on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT and TGACT.

8. METHOD, according to any one of claims 1 to 7, characterized in that the substitution of one or more nucleotides in the DRACH motif is performed using one or more codon usage guidelines selected from the group consisting of: (a) never using the GAC codon to encode aspartic acid when eliminating the DRACH motif; (b) never using the GAA codon to encode glutamic acid when eliminating the DRACH motif; (c) never using the GGA codon to encode glycine when eliminating the DRACH motif; (d) never using the AAA codon to encode lysine when eliminating the DRACH motif; (e) never using the AAC codon to encode asparagine when eliminating the DRACH motif; and / or (f) always using the ACG codon to encode threonine when eliminating the DRACH motif.

9. METHOD, according to any one of claims 1 to 8, characterized in that the codon optimization further comprises implementing, by the total polynucleotide sequence, one or more codon usage guidelines selected from the group consisting of: (a) never using the codon GAC to encode aspartic acid by the total polynucleotide sequence; (b) never using the codon GAA to encode glutamic acid by the total polynucleotide sequence; (c) never using the codon GGA to encode glycine by the total polynucleotide sequence; (d) never using the codon AAA to encode lysine by the total polynucleotide sequence; (e) never using the codon AAC to encode asparagine by the total polynucleotide sequence; and / or (f) never using the codon ACG to encode threonine by the total polynucleotide sequence.

10. METHOD, according to any one of claims 1 to 9, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides which together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism than the frequency with which the unmodified codon occurs in the target organism.

11. METHOD, according to any one of claims 1 to 10, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism.

12. METHOD, according to claim 10 or 11, characterized in that the target organism is a mammal, optionally wherein the mammal is a human.

13. METHOD, according to any one of claims 1 to 12, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

14. METHOD, according to any one of claims 1 to 13, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by modulating the guanine-cytosine (GC) content of the polynucleotide, secondary structure of the polynucleotide, mRNA motifs, ribosomal binding sites and / or translation rates.

15. METHOD, according to claim 14, characterized in that the GC content of the polynucleotide is adjusted to between 50% and 80%. Petition 870250096842, dated 10 / 23 / 2025, page 133 / 180 5 / 27 16. METHOD, according to claim 15, characterized in that the GC content of the polynucleotide is adjusted to between 65% and 75%.

17. METHOD, according to any one of claims 1 to 16, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method further comprises replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides which together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism relative to the frequency with which the unmodified codon occurs in the target organism.

18. METHOD, according to any one of claims 1 to 16, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method further comprises replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism.

19. METHOD, according to claim 17 or 18, characterized in that the target organism is a mammal, optionally wherein the mammal is a human.

20. METHOD, according to any one of claims 1 to 19, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method further comprises optimizing by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

21. METHOD, according to any of the claims Petition 870250096842, of 10 / 23 / 2025, pp. 134 / 180 6 / 27 1 to 20, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method further comprises modulating the GC content, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

22. METHOD, according to claim 21, characterized in that the GC content of the polynucleotide is adjusted to between 50% and 80%.

23. METHOD, according to claim 22, characterized in that the GC content of the polynucleotide is adjusted to between 65% and 75%.

24. METHOD, according to any one of claims 1 to 23, characterized in that the amino acid sequence of the protein or polypeptide shares at least 85% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

25. METHOD, according to any one of claims 1 to 24, characterized in that the amino acid sequence of the protein or polypeptide shares at least 100% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

26. METHOD, according to any one of claims 1 to 25, characterized in that the number of DRACH motifs in the polynucleotide sequence is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20% or at least 10%, compared with a corresponding polynucleotide sequence that has not been subjected to the method.

27. METHOD, according to claim 26, characterized in that the number of DRACH motifs in the polynucleotide sequence is reduced by at least 100% compared with a corresponding polynucleotide sequence that has not been subjected to the method.

28. METHOD, according to any one of claims 1 to 27, characterized in that the codon optimization method results in high mRNA transcript stability or half-life.

29. METHOD, according to claim 28, characterized in that the high stability of the mRNA transcript or half-life is assessed by an increase in concentration or relative abundance compared to a corresponding polynucleotide sequence that has not been subjected to the method.

30. METHOD, according to claim 29, characterized in that the elevation in concentration or relative abundance of the mRNA transcript compared to the mRNA transcript of the native polynucleotide sequence is measured by RT-PCR, fluorimetry, colorimetry, spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses or a matrix using one or more oligonucleotides or hybridizing antibodies against the mRNA transcript of interest.

31. METHOD, according to claim 28, characterized in that the high stability of the mRNA transcript or half-life is assessed by reduced decay rates, as detected by one or more pulse-chase methodologies.

32. METHOD, according to claims 1 to 31, characterized in that reducing the number of DRACH motifs in the polynucleotide sequence reduces the number of N(6)-methyladenosine (m6A) modifications in an mRNA transcript compared with a corresponding polynucleotide sequence that has not been subjected to the method.

33. METHOD, according to any one of claims 1 to 32, characterized in that the codon optimization method results in high protein expression or stability compared to the protein encoded by a corresponding polynucleotide sequence that has not been subjected to the method.

34. METHOD, according to any one of claims 26 to 33, characterized in that the corresponding polynucleotide sequence is a wild-type polynucleotide sequence.

35. METHOD, according to claim 33 or 34, characterized in that the expression or stability of the protein is elevated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% compared to a corresponding polynucleotide sequence that has not been subjected to the method.

36. METHOD, according to any one of claims 33 to 35, characterized in that the expression or stability of the protein is elevated by about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times or more than 50 times compared with a corresponding polynucleotide sequence that has not been subjected to the method.

37. METHOD, according to any one of claims 33 to 36, characterized in that the elevation in protein expression is measured by mass spectrometry, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation methods, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses or an array using aptamers or antibodies against the protein of interest.

38. POLYNUCLEOTIDE, characterized in that it is produced by the method of any one of claims 1 to 37. Petition 870250096842, dated 10 / 23 / 2025, pp. 137 / 180 9 / 27 39. METHOD FOR DISTRIBUTING A POLYNUCLEOTIDE SEQUENCE encoding a polypeptide, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) providing the resulting polynucleotide sequence to the host cell.

40. METHOD FOR EXPRESSING AN mRNA TRANSCRIPT in a host cell from a polynucleotide sequence encoding a polypeptide, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) providing the polynucleotide sequence to the host cell.

41. METHOD FOR EXPRESSING A PROTEIN in a host cell from a polynucleotide sequence encoding the protein, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded protein and (ii) providing the polynucleotide sequence to the host cell.

42. METHOD FOR DISTRIBUTING A POLYNUCLEOTIDE SEQUENCE encoding a polypeptide to a subject, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) providing the resulting polynucleotide sequence to the subject.

43. METHOD FOR EXPRESSING AN mRNA TRANSCRIPT in a subject from a polynucleotide sequence encoding a polypeptide, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) providing the polynucleotide sequence to the subject.

44. METHOD FOR EXPRESSING A PROTEIN in a subject from a polynucleotide sequence encoding the protein, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within the polynucleotide sequence by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded protein and (ii) providing the polynucleotide sequence to the subject.

45. METHOD, according to any one of claims 39 to 44, characterized in that the substitution is carried out on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, TGACT, AAACA, AAACC, AAACT, AGACC, GAACA, GGACC, TAACA, TAACC, TAACT, TGACA and TGACC.

46. ​​METHOD, according to claim 45, characterized in that the substitution is carried out on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, and TGACT.

47. METHOD, according to any one of claims 39 to 46, characterized in that the substitution of one or more nucleotides in the DRACH motif is performed using one or more codon usage guidelines selected from the group consisting of: (a) never using the GAC codon to encode aspartic acid when eliminating the DRACH motif; (b) never using the GAA codon to encode glutamic acid when eliminating the DRACH motif; (c) never using the GGA codon to encode glycine when eliminating the DRACH motif; (d) never using the AAA codon to encode lysine when eliminating the DRACH motif; (e) never using the AAC codon to encode asparagine when eliminating the DRACH motif; and / or (f) always using the ACG codon to encode threonine when eliminating the DRACH motif.

48. METHOD, according to any one of claims 39 to 47, characterized in that the codon optimization further comprises implementing, by the total polynucleotide sequence, one or more codon usage guidelines selected from the group consisting of: (a) never using the codon GAC for aspartic acid by the total polynucleotide sequence; (b) never using the codon GAA for glutamic acid by the total polynucleotide sequence; (c) never using the codon GGA for glycine by the total polynucleotide sequence; (d) never using the codon AAA for lysine by the total polynucleotide sequence; (e) never using the codon AAC for asparagine by the total polynucleotide sequence; and / or Petition 870250096842, of 10 / 23 / 2025, p. 140 / 180 12 / 27 (f) never use the ACG codon for threonine by the total polynucleotide sequence.

49. METHOD, according to any one of claims 39 to 48, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides which together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism than the frequency with which the unmodified codon occurs in the target organism.

50. METHOD, according to any one of claims 39 to 49, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism.

51. METHOD, according to claim 49 or 50, characterized in that the target organism is a mammal, optionally wherein the mammal is a human.

52. METHOD, according to any one of claims 39 to 51, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

53. METHOD, according to any one of claims 39 to 52, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by modulating the polynucleotide GC content, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

54. METHOD, according to claim 53, characterized in that the GC content of the polynucleotide is adjusted to between 50% and 80%.

55. METHOD, according to claim 54, characterized in that the GC content of the polynucleotide is adjusted to between 65% and 75%.

56. METHOD, according to any one of claims 39 to 55, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises substituting one or more nucleotides in a codon by an equivalent number of alternative nucleotides which together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism relative to the frequency with which the unmodified codon occurs in the target organism.

57. METHOD, according to any one of claims 39 to 56, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises substituting one or more codons in the polynucleotide sequence by an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism.

58. METHOD, according to claim 56 or 57, characterized in that the target organism is a mammal, optionally wherein the mammal is a human. Petition 870250096842, dated 10 / 23 / 2025, pp. 142 / 180 14 / 27 59. METHOD, according to any one of claims 39 to 58, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises optimizing by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network or a combination thereof.

60. METHOD, according to any one of claims 39 to 59, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the codon optimization method further comprises modulating the GC content, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

61. METHOD, according to claim 60, characterized in that the GC content of the polynucleotide is adjusted to between 50% and 80%.

62. METHOD, according to claim 61, characterized in that the GC content of the polynucleotide is adjusted to between 65% and 75%.

63. METHOD, according to any one of claims 39 to 62, characterized in that the amino acid sequence of the protein or polypeptide shares at least 85% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

64. METHOD, according to any one of claims 39 to 63, characterized in that the amino acid sequence of the protein or polypeptide shares at least 100% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

65. METHOD, according to any of the claims Petition 870250096842, of 10 / 23 / 2025, pp. 143 / 180 15 / 27 39 to 64, characterized in that the number of DRACH motifs in the polynucleotide sequence is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20% or at least 10%, compared to a corresponding polynucleotide sequence that has not been subjected to the method.

66. METHOD, according to claim 65, characterized in that the number of DRACH motifs in the polynucleotide sequence is reduced by at least 100% compared with a corresponding polynucleotide sequence that has not been subjected to the method.

67. METHOD, according to any one of claims 39 to 66, characterized in that the method results in high stability of the mRNA transcript or half-life.

68. METHOD, according to claim 67, characterized in that the high stability of the mRNA transcript or half-life is assessed by an increase in concentration or relative abundance compared to a corresponding polynucleotide sequence that has not been subjected to the method.

69. METHOD, according to claim 68, characterized in that the elevation in concentration or relative abundance of the mRNA transcript compared with the mRNA transcript of the native polynucleotide sequence is measured by RT-PCR, fluorimetry, colorimetry, spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses or a matrix using one or more oligonucleotides or hybridizing antibodies against the mRNA transcript of interest.

70. METHOD, according to claim 67, characterized in that the high stability of the mRNA transcript or half-life is assessed by reduced decay rates, as detected by one or more pulse-chase methodologies. Petition 870250096842, dated 10 / 23 / 2025, pp. 144 / 180 16 / 27 71. METHOD, according to any one of claims 39 to 70, characterized in that reducing the number of DRACH motifs in the polynucleotide sequence reduces the number of N(6)methyladenosine (m6A) modifications in an mRNA transcript compared with a corresponding polynucleotide sequence that has not been subjected to the method.

72. METHOD, according to any one of claims 39 to 71, characterized in that the method results in high protein expression or stability compared to the protein encoded by a corresponding polynucleotide sequence that has not been subjected to the method.

73. METHOD, according to any one of claims 65 to 72, characterized in that the corresponding polynucleotide sequence is a wild-type polynucleotide sequence.

74. METHOD, according to claim 72 or 73, characterized in that the expression or stability of the protein is elevated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% compared with a corresponding polynucleotide sequence that has not been subjected to the method.

75. METHOD, according to any one of claims 72 to 74, characterized in that the expression or stability of the protein is elevated by about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times or more than 50 times compared with a corresponding polynucleotide sequence that has not been subjected to the method.

76. METHOD, according to any one of claims 72 to 75, characterized in that the elevation in protein expression is measured by mass spectrometry, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation methods, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses or a matrix using aptamers or antibodies against the protein of interest.

77. METHOD, according to any one of claims 39 to 41 and 45 to 76, characterized in that the polynucleotide is delivered to the host cell by contacting the host cell with a vehicle comprising the polynucleotide.

78. METHOD, according to any one of claims 42 to 76, characterized in that the polynucleotide is delivered to the subject by administering to the subject a vehicle comprising the polynucleotide.

79. METHOD, according to claim 77 or 78, characterized in that the vehicle is selected from the group consisting of a lipid nanoparticle, a liposome, a viral genome and a viral vector.

80. METHOD, according to claim 79, characterized in that the viral vector is an adeno-associated virus (AAV), an adenovirus or a lentivirus.

81. METHOD, according to any one of claims 77 to 80, characterized in that the viral vector is an AAV.

82. METHOD, according to claim 81, characterized in that the AAV is pseudotyped.

83. METHOD, according to claim 81 or 82, characterized in that the AAV comprises an inverted terminal repeat (ITR) sequence and a capsid protein derived from different serotypes.

84. METHOD, according to any one of claims 39 to 41 and 45 to 83, characterized in that the delivery to the host cell occurs in vivo, in vitro or ex vivo. Petition 870250096842, dated 10 / 23 / 2025, pp. 146 / 180 18 / 27 85. METHOD, according to any one of claims 42 to 83, characterized in that delivery to the subject occurs by means of (i) in vivo administration of the polynucleotide to the subject or (ii) ex vivo expression of the polynucleotide in a host cell, followed by administration of the host cell to the subject.

86. METHOD, according to any one of claims 1 to 85, characterized in that the method is used to treat a subject who has or is at risk of having a disease represented by low expression or activity of a protein.

87. METHOD FOR TREATING A SUBJECT WHO HAS OR IS AT RISK OF DEVELOPING A DISEASE, the method characterized in that it comprises (i) replacing one or more nucleotides in a DRACH motif within a polynucleotide sequence encoding a disease-associated polynucleotide with an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide and (ii) administering the polynucleotide sequence to the subject.

88. METHOD FOR TREATING A SUBJECT WHO HAS OR IS AT RISK OF DEVELOPING A DISEASE, the method characterized in that it comprises administering a polynucleotide sequence encoding a disease-associated polypeptide to the subject, wherein prior to administration, one or more nucleotides in a DRACH motif within the polynucleotide sequence have been replaced by an equivalent number of alternative nucleotides that eliminate the DRACH motif, wherein the substitution does not alter the amino acid sequence of the encoded polypeptide.

89. METHOD, according to claims 87 to 88, characterized in that the substitution is carried out on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, Petition 870250096842, dated 10 / 23 / 2025, page 147 / 180 19 / 27 GGACA, GGACT, TGACT, AAACA, AAACC, AAACT, AGACC, GAACA, GGACC, TAACA, TAACC, TAACT, TGACA and TGACC.

90. METHOD, according to claim 89, characterized in that the substitution is carried out on a DRACH motif selected from the group consisting of AGACA, AGACT, GAACC, GAACT, GGACA, GGACT, and TGACT.

91. METHOD, according to any one of claims 87 to 90, characterized in that the substitution of one or more nucleotides in the DRACH motif is performed using one or more codon usage guidelines selected from the group consisting of: (a) never using the GAC codon to encode aspartic acid when eliminating the DRACH motif; (b) never using the GAA codon to encode glutamic acid when eliminating the DRACH motif; (c) never using the GGA codon to encode glycine when eliminating the DRACH motif; (d) never using the AAA codon to encode lysine when eliminating the DRACH motif; (e) never using the AAC codon to encode asparagine when eliminating the DRACH motif; and / or (f) always using the ACG codon to encode threonine when eliminating the DRACH motif.

92. METHOD, according to any one of claims 87 to 91, characterized in that prior to administration, the polynucleotide sequence has been codon-optimized by a method comprising implementing, by the total polynucleotide sequence, one or more codon usage guidelines selected from the group consisting of: (a) never using the codon GAC to encode aspartic acid by the total polynucleotide sequence; (b) never using the codon GAA to encode glutamic acid by the total polynucleotide sequence; (c) never using the codon GGA to encode glycine by the total polynucleotide sequence; (d) never using the codon AAA to encode lysine by the total polynucleotide sequence; (e) never use the AAC codon to encode asparagine by the full polynucleotide sequence; and / or (f) never use the ACG codon to encode threonine by the full polynucleotide sequence.

93. METHOD, according to any one of claims 87 to 92, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more nucleotides in a codon with an equivalent number of alternative nucleotides which together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism than the frequency with which the unmodified codon occurs in the target organism.

94. METHOD, according to any one of claims 87 to 93, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence has been codon-optimized by replacing one or more codons in the polynucleotide sequence with an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism.

95. METHOD, according to claim 93 or 94, characterized in that the target organism is a mammal, optionally Petition 870250096842, of 10 / 23 / 2025, pp. 149 / 180 21 / 27 where the mammal is a human.

96. METHOD, according to any one of claims 87 to 95, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network, or a combination thereof.

97. METHOD, according to any one of claims 87 to 98, characterized in that, prior to the substitution of one or more nucleotides in the DRACH motif, the polynucleotide sequence was codon-optimized by modulating the guanine-cytosine (GC) content of the polynucleotide, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

98. METHOD, according to claim 97, characterized in that the GC content of the polynucleotide is adjusted to between 50% and 80%.

99. METHOD, according to claim 98, characterized in that the GC content of the polynucleotide is adjusted to between 65% and 75%.

100. METHOD, according to any one of claims 87 to 99, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises substituting one or more nucleotides in a codon by an equivalent number of alternative nucleotides which together comprise a synonymous codon that occurs naturally at a higher frequency in a target organism relative to the frequency with which the unmodified codon occurs in the target organism.

101. METHOD, according to any of the claims Petition 870250096842, dated 10 / 23 / 2025, pp. 150 / 180 22 / 27 87 to 100, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises substituting one or more codons in the polynucleotide sequence by an equivalent number of synonymous codons that occur naturally at a higher frequency in a target organism relative to the frequency with which the one or more unmodified codons occur in the target organism.

102. METHOD, according to claim 100 or 101, characterized in that the target organism is a mammal, optionally wherein the mammal is a human.

103. METHOD, according to any one of claims 87 to 102, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises optimizing by means of a codon optimization web tool, codon optimization software, quantum computing, a heuristic scoring method, a neural network or a combination thereof.

104. METHOD, according to any one of claims 87 to 103, characterized in that, after the substitution of one or more nucleotides in the DRACH motif, the method further comprises modulating GC content, polynucleotide secondary structure, mRNA motifs, ribosomal binding sites and / or translation rates.

105. METHOD, according to claim 104, characterized in that the GC content of the polynucleotide is adjusted to between 50% and 80%.

106. METHOD, according to claim 105, characterized in that the GC content of the polynucleotide is adjusted to between 65% and 75%.

107. METHOD, according to any one of claims 87 to 106, characterized in that the amino acid sequence of the protein or polypeptide shares at least 85% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

108. METHOD, according to any one of claims 87 to 107, characterized in that the amino acid sequence of the protein or polypeptide shares at least 100% sequence identity with a wild-type amino acid sequence of the protein or polypeptide following codon optimization.

109. METHOD, according to any one of claims 87 to 108, characterized in that the number of DRACH motifs in the polynucleotide sequence is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20% or at least 10%, compared with a corresponding polynucleotide sequence that has not been subjected to the method.

110. METHOD, according to claim 109, characterized in that the number of DRACH motifs in the polynucleotide sequence is reduced by at least 100% compared with a corresponding polynucleotide sequence that has not been subjected to the method.

111. METHOD, according to any one of claims 87 to 110, characterized in that the method results in high stability of the mRNA transcript or half-life.

112. METHOD, according to claim 111, characterized in that the high stability of the mRNA transcript or half-life is assessed by an increase in concentration or relative abundance compared to a reference.

113. METHOD, according to claim 112, characterized in that the elevation in concentration or relative abundance of the mRNA transcript compared with the mRNA transcript of the native polynucleotide sequence is measured by RT-PCR, fluorimetry, colorimetry, spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses or a matrix using one or more oligonucleotides or hybridizing antibodies against the mRNA transcript of interest.

114. METHOD, according to claims 87 to 113, characterized in that reducing the number of DRACH motifs in the polynucleotide sequence reduces the number of N(6)-methyladenosine (m6A) modifications in an mRNA transcript compared with a corresponding polynucleotide sequence that has not been subjected to the method.

115. METHOD, according to any one of claims 87 to 114, characterized in that the method results in high protein expression or stability compared to a reference.

116. METHOD, according to any one of claims 109 to 115, characterized in that the corresponding polynucleotide sequence is a wild-type polynucleotide sequence.

117. METHOD, according to any one of claims 112 to 116, characterized in that the reference is a sample obtained from a subject who has not received treatment or a sample obtained from the subject before treatment.

118. METHOD, according to any one of claims 115 to 117, characterized in that the expression or stability of the protein is elevated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% compared with a corresponding polynucleotide sequence that has not been subjected to the method.

119. METHOD, according to any of the claims Petition 870250096842, dated 10 / 23 / 2025, pp. 153 / 180 25 / 27 115 to 118, characterized in that the expression or stability of the protein is elevated by about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times or more than 50 times compared to a corresponding polynucleotide sequence that has not been subjected to the method.

120. METHOD, according to any one of claims 115 to 119, characterized in that the elevation in protein expression is measured by mass spectrometry, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation methods, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses or an array using aptamers or antibodies against the protein of interest.

121. METHOD, according to any one of claims 87 to 120, characterized in that the polynucleotide is administered to the subject by contacting the subject with a vehicle comprising the polynucleotide.

122. METHOD, according to claim 121, characterized in that the vehicle is selected from a lipid nanoparticle, a liposome, a viral genome and a viral vector.

123. METHOD, according to claim 122, characterized in that the viral vector is an adeno-associated virus (AAV), an adenovirus, a retrovirus, a lentivirus, or a double-stranded DNA virus.

124. METHOD, according to claim 122 or 123, characterized in that the viral vector is an AAV.

125. METHOD, according to claim 124, characterized in that the AAV is pseudotyped.

126. METHOD, according to claim 124 or 125, characterized in that the AAV comprises an inverted terminal repeat (ITR) sequence and a capsid protein derived from different serotypes.

127. METHOD, according to any one of claims 87 to 126, characterized in that the administration takes place by directly supplying the subject, in vivo, with the polynucleotide or by expressing the polynucleotide in a host cell ex vivo and subsequently administering the host cell to the subject.

128. METHOD, according to any one of claims 87 to 127, characterized in that the distribution is carried out by intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intra-arterial, intraventricular, intranasal, intraorbital, intracranial or intraosseous administration.

129. METHOD, according to any one of claims 87 to 128, characterized in that the treatment is a monotherapy.

130. METHOD, according to any one of claims 87 to 128, characterized in that the method is combined with the administration of one or more additional therapeutic agents.

131. KIT, characterized in that it comprises a leaflet instructing a user to perform a codon optimization method comprising modifying a polynucleotide sequence encoding a polypeptide by replacing one or more nucleotides with a DRACH motif in the polynucleotide sequence without altering the amino acid sequence of the encoded polypeptide, thereby eliminating one or more DRACH motifs.

132. KIT, characterized in that it comprises a leaflet instructing the user to perform the method of any of claims 1 to 37 and 39 to 130.

133. KIT, according to claim 131 or 132, characterized in that the kit comprises a device for administering the polynucleotide sequence to a subject.

134. A KIT, according to any one of claims 131 to 133, characterized in that the kit comprises one or more binding molecules for detecting the expression of an mRNA transcript encoding the polypeptide or the expression or activity of the polypeptide. Petition 870250096842, dated 10 / 23 / 2025, pp. 156 / 180

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