A critical analysis of codon optimization in human therapeutics

Abstract

Codon optimization describes gene engineering approaches that use synonymous codon changes to increase protein production. Applications for codon optimization include recombinant protein drugs and nucleic acid therapies, including gene therapy, mRNA therapy, and DNA/RNA vaccines. However, recent reports indicate that codon optimization can affect protein conformation and function, increase immunogenicity, and reduce efficacy. We critically review this subject, identifying additional potential hazards including some unique to nucleic acid therapies. This analysis highlights the evolved complexity of codon usage and challenges the scientific bases for codon optimization. Consequently, codon optimization may not provide the optimal strategy for increasing protein production and may decrease the safety and efficacy of biotech therapeutics. We suggest that the use of this approach is reconsidered, particularly for in vivo applications.

Keywords: A-to-I editing; codon optimization; gene therapy; mRNA therapy; tRNA wobble; vaccine.

Figure 1. The degenerate genetic code

The circular representation of the genetic code indicates wobble, tRNA gene presence, and codon usage in humans. Amino acids are indicated in the outside yellow ring, using the one letter notation. Stop codons are indicated by a dash. The 1^st, 2^nd, and 3^rd nucleotides of codons are indicated in the inner, middle, and outer nucleotide circles, respectively. Codons that lack a corresponding tRNA gene in humans are indicated by red bars [17]. For illustration, potential wobble codons are indicated, based on Crick’s wobble rules. G-U wobble base pairing codons are indicated by dark blue bars. Potential U-G wobble base pairing codons are indicated by dark blue striped bars. Codons capable of I-U and I-C base pairing are indicated by light blue bars. Potential I-A wobble base pairing codons are indicated by light blue striped bars. Note that 7 possible inosine modifications have been reported for yeast and 8 for mammalian tRNAs [18, 19]. Codons that recognize tRNAs with other modifications that may extend or restrict wobble are not indicated [20, 123]. The frequency of codon usage in human is indicated by the grey bars. The density of the bars corresponds to codon usage ranging from 0% usage (white) to 100% usage (black). Human codon usage data is from the “Codon Usage Database” (http://www.kazusa.or.jp/codon/).

Figure 2. tRNA-mRNA base pairing and wobble base pairing

(A) Nucleotide positions. tRNA numbering is according to [124]. Structural features in the tRNA are indicated; the anticodon occupies nucleotide positions 34–36. Conserved nucleotide positions are highlighted in black. Base pairing of a tRNA to a codon in mRNA is indicated, with the 1^st, 2^nd, and 3^rd positions of the codons labeled 1, 2, 3. Different colors represent different codons. Wobble base pairing occurs between position 34 of the tRNA and the 3^rd position of the codon, indicated in red. (B) A tRNA with a U or G at position 34 can Watson-Crick base pair and wobble base pair as shown. Only the anti-codon loop of the tRNAs is shown in panels B-D. (C) A tRNA with I at position 34 can wobble base pair to U, C, or A as shown. (D) Superwobbling. A tRNA with an unmodified U in tRNA position 34 can base pair to C and wobble base pair to G, A, and U. To date, superwobbling has only been reported in chloroplasts [21, 22].

Figure 3. Codon frequencies in human

(A) The frequency of occurrence of amino acids (AA). AAs are listed on the abscissa in one letter notation. The frequency with which each AA is encoded is listed as a percentage on the ordinate. (B) Normalized frequency of occurrence of AAs. For each amino acid, the amino acid frequency (%) is normalized to the number of synonymous codons. (C) The frequency of occurrence of codons. Codons are listed on the abscissa of the bar graph; in each case, the frequency of usage (as a percentage) is plotted on the ordinate. The codons are ordered according to their reported frequency of occurrence. Stop codons are not listed. White bars indicate codons that can be decoded only by corresponding cognate tRNAs. Black bars indicate codons that lack a cognate tRNA gene and can be decoded only by wobble tRNAs. Grey bars indicate codons that can be decoded by both cognate and wobble tRNAs. Note that potential U-G and I-A wobble interactions are not considered in this bar graph as these wobble interactions do not yet appear to have been confirmed in human. (D) Normalized codon frequency. The codon frequency (%) has been normalized by dividing the codon frequency by the average number of cognate and wobble tRNA genes for each codon. The human codon usage data is from “Codon Usage Database” (http://www.kazusa.or.jp/codon/).

Figure 4. Translation of full-length protein and cryptic peptides

(A) Translation from a natural cap-dependent mRNA. Schematic represents an mRNA that initiates translation from multiple start sites, including AUG and noncanonical start sites. A ribosomal complex assembled at the 5′ m7G cap-structure of an mRNA is indicated. The 40S subunit is tethered to the mRNA via the eIF4F complex of initiation factors and initiation factor eIF3, indicated as green circles [81]. The 40S subunit is bound to the ternary complex, which contains initiation factor eIF2, indicated in blue, GTP, and the initiator Met-tRNA, indicated as a cloverleaf structure. The arrows indicate possible start sites for translation initiation. Start sites include AUG and non-canonical start sites such as CUG, ACG, and GUG [125]. Protein synthesis starting at the initiation codon gives rise to full-length protein, which is represented by the long blue bar. Shorter peptides initiating from alternative start sites in the same reading frame are indicated by the shorter blue bars. Peptides initiating from out-of-frame start sites will generate peptides with different amino acid sequences; these are indicated by the grey and green bars, which represent the two alternative (out-of-frame) reading frames. (B) Translation from a codon-optimized mRNA. In this case, the full-length protein and any in-frame peptides will be the same as those from the natural mRNA. Some in-frame peptides may be lost if a non-AUG codon is modified (e.g. the peptide arising from the internal GUG). In addition, most if not all of the out-of-frame peptides will be lost and a new set of out-of-frame peptides will be encoded. These new peptides from the two new alternative reading frames are indicated in fuchsia and dark orange.