METHODS FOR REDUCING RNA IMMUNOGENICITY AND RNA MOLECULES WITH DECREASED IMMUNOGENICITY
20230295637 · 2023-09-21
Assignee
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
C12N15/67
CHEMISTRY; METALLURGY
C12P19/34
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a method for decreasing the immunogenicity of an RNA molecule and/or at least maintaining the translation efficacy thereof. The present invention further relates to an RNA molecule, which is modified as compared to a corresponding wildtype RNA molecule, wherein the exchange of codons results in the total cytidine content of the modified RNA molecule being at least 10% less than the total cytidine content of the corresponding RNA molecule transcribed from said wildtype DNA sequence. The present invention also relates to an RNA molecule, wherein the exchange of codons results in the total uridine content of the modified RNA molecule being at least 10% less than the total uridine content of the corresponding RNA molecule transcribed from said wild-type DNA sequence. Finally, the present invention relates to the use of an RNA molecule of this invention in genome editing.
Claims
1-37. (canceled)
38. A method for decreasing the immunogenicity of an RNA molecule and/or at least maintaining the translation efficacy thereof, the method comprising: a) providing a wildtype DNA sequence as a template for RNA transcription; b) selecting from the DNA sequence the coding sequence of the sense DNA strand, which comprises the sequence from the ATG codon to the first in-frame stop codon; c) dividing the coding sequence into codons; d) exchanging one or more codons that comprise one or more cytidine nucleotides for an available alternative codon comprising less cytidine nucleotides and resulting in a similar amino acid to obtain a DNA molecule with a modified DNA sequence; and e) producing a modified RNA molecule from the DNA molecule with the modified DNA sequence, wherein the exchange of codons results in the total cytidine content of the modified RNA molecule being at least 10% less than the total cytidine content of the corresponding RNA molecule transcribed from said wildtype DNA sequence.
39. The method of claim 38, further comprising repeating step d) with codons comprising thymidine nucleotides before producing the modified RNA molecule, wherein the exchange of codons results in the total uridine content of the modified RNA molecule being at least 10% less than the total uridine content of the corresponding RNA molecule transcribed from said wild-type DNA sequence.
40. The method of claim 38, wherein the alternative codon encodes the same amino acid.
41. The method of claim 38, wherein the cytidine nucleotides and the thymidine nucleotides in a codon are replaced with another non-modified nucleotide, in particular a guanosine or adenosine nucleotide.
42. The method of claim 38, wherein codons are exchanged in a random fashion, or in the order of their appearance in the coding sequence.
43. The method of claim 38, wherein codons are exchanged with alternative codons that occur with the highest frequency in the human genome.
44. The method of claim 38, wherein the available alternative codon comprising less cytidine nucleotides encodes the same amino acid, or wherein the available alternative codon comprising less cytidine nucleotides result in conservative replacement of the encoded amino acid.
45. The method of claim 38, wherein the codons are exchanged according to any one of the codon exchange tables 1A, 1B, 2A, 2B, 2C, 2D.
46. An RNA molecule, which is modified as compared to a corresponding wildtype RNA molecule, wherein the modification comprises a reduction of cytidine nucleotides to the extent that the exchange of codons results in the total cytidine content being at least 10% less than the total cytidine content of the corresponding RNA molecule transcribed from said wild-type DNA sequence, wherein the modification optionally further comprises a reduction of uridine nucleotides to the extent that the exchange of codons results in the total uridine content being at least 10% less than the total uridine content of the corresponding RNA molecule transcribed from said wild-type DNA sequence, wherein the modified RNA molecule is in particular less immunogenic than the wildtype RNA molecule and/or upon translation results in a similar or higher protein production, in particular a significantly higher protein production than the wildtype RNA molecule.
47. The RNA molecule of claim 46, which is a long non-coding RNA or a messenger RNA molecule (mRNA) encoding a peptide, polypeptide or protein.
48. The RNA molecule of claim 46, wherein the nucleotides replacing the cytidines or uridines of the wild type RNA molecule in the modified RNA molecule are non-modified nucleotides, wherein the RNA molecule optionally comprises a modification as compared to a wildtype RNA molecule, which modification is a deletion and/or substitution of one or more of the cytidine nucleotides, in particular a substitution or deletion of one or more of the cytidine and optionally uridine nucleotides from an untranslated region of the RNA molecule.
49. The RNA molecule of claim 48, which is an mRNA and wherein the amino acid sequence of the peptide, polypeptide or protein encoded by the modified mRNA molecule is the same as the amino acid sequence of the polypeptide or protein encoded by the wildtype mRNA molecule, or is different from the amino acid sequence of the peptide, polypeptide or protein encoded by the wildtype mRNA molecule, which difference between the amino acid sequence encoded by the modified RNA sequence compared to the amino acid sequence encoded by the wildtype RNA sequence is less than 1/200 codons.
50. The RNA molecule of claim 46, wherein the RNA sequence is modified by substituting cytidine and optionally uridine nucleotides by adenosine or guanosine nucleotides, in particular non-modified adenosine or guanosine nucleotides.
51. The RNA molecule of claim 46, which is an mRNA and wherein the cytidine content and optionally the uridine content is reduced in the coding region of the mRNA, and/or wherein the cytidine content and optionally the uridine content is reduced in the non-coding region of the mRNA, in particular in the 5′UTR region and/or 3′UTR region.
52. The RNA molecule of claim 46, wherein in order of increased preference at least 10, 15, 20, 25, 30, 35, 40, 45, 50% of the cytidine and optionally uridine nucleotides of the RNA sequence of the wildtype RNA molecule are replaced by a nucleotide that is not cytidine or uridine, respectively, or deleted.
53. The RNA molecule of claim 48 for use in therapy, wherein the therapy is selected from replacement of absent and/or defective polypeptides or proteins having a biological activity, supplementation of an endogenous protein to enhance cellular processes counteracting a disorder or repress cellular processes causing a disorder, introduction of non-endogenous biologically active proteins in a patient, wherein the therapy is in particular for treatment of disorders that involve inflammation, in particular chronic kidney disease, focal segmental glomerulosclerosis, lupus nephritis, glomerulonephritis, membranoproliferative glomerulonephritis, interstitial nephritis, IgA nephropathy (Berger's disease), pyelonephritis, Goodpasture's syndrome, Wegener's granulomatosis, acute kidney disease, kidney transplant rejection, inflammatory bowel disease, ulcerative colitis, Crohn's disease, coeliac disease, atopic dermatitis, psoriasis, eczema, Behçet's disease, acne, pyoderma, rosacea, systemic lupus erythematosus, asthma, chronic obstructive pulmonary disease, COPD, pneumonitis rheumatoid arthritis, periodontitis, sinusitis, transplant rejection, ischemia reperfusion injury (also known as reperfusion injury), atherosclerosis, vasculitis, inflammatory cornea disorders, diabetic nephropathy, sepsis, liver fibrosis/cirrhosis, or for use in diagnosis, wherein the diagnosis is selected from detecting specific cells, detecting the presence or absence of proteins, in particular tumor suppressor proteins, proteins signaling inflammation, fibrosis and/or cell-stress, or for use in prophylaxis, wherein the RNA molecule is used as a vaccine, in particular a vaccine against viruses, such as influenza viruses or corona viruses.
54. The RNA molecule of claim 46, obtainable or produced by a method comprising: a) providing a wildtype DNA sequence as a template for RNA transcription; b) selecting from the DNA sequence the coding sequence of the sense DNA strand, which comprises the sequence from the ATG codon to the first in-frame stop codon; c) dividing the coding sequence into codons; d) exchanging one or more codons that comprise one or more cytidine nucleotides for an available alternative codon comprising less cytidine nucleotides and resulting in a similar amino acid to obtain a DNA molecule with a modified DNA sequence; and e) producing a modified RNA molecule from the DNA molecule with the modified DNA sequence, wherein the exchange of codons results in the total cytidine content of the modified RNA molecule being at least 10% less than the total cytidine content of the corresponding RNA molecule transcribed from said wildtype DNA sequence.
55. A pharmaceutical composition comprising the modified RNA molecule of claim 46.
56. A use of the RNA molecule of claim 46 in genome editing, wherein the RNA molecule is for encoding an RNA-guided endonuclease and/or a guide RNA in CRISPR technology.
Description
[0081] In this application reference is made to the following figures:
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[0093] The present invention will be further illustrated in the Example that follows.
EXAMPLES
Example 1
Sequence-Engineering of mRNAs According to the Invention
[0094] To obtain an mRNA according to the invention, first the wildtype DNA sequence of the gene of interest is obtained from sources known to a person skilled in the art. Next, the coding sequence is isolated by identification of the start-codon and in-frame stop-codon according to information from literature, provided by the manufacturer or other methodologies known to a person skilled in the art. For secreted nanoluciferase the coding sequence (Coding sequence 1) was obtained from the manufacturer (Promega). For murine Erythropoietin (mEPO), the coding sequence (Coding sequence 2) was obtained from NCBI (NCBI Reference Sequence: NM_007942.2). For enhanced green fluorescent protein (eGFP), the sequence was previously developed in-house based on literature (Coding sequence 3).
[0095] Next, the coding sequence was modified according to the invention. For this, the coding sequence was divided in codons according to methods known to the person skilled in the art. Next, each codon identified in the WT-sequence present in the column named ‘Original codon’ was exchanged with the corresponding codon from the column named ‘Swap codon’ from the corresponding codon exchange table. For codons not present in the column name ‘Original codon’ no changes were made. In this study, for the U-depleted variants of secNLuc, mEPO and eGFP, codon exchange table 4A was used. In this study, for the UC-depleted variants of secNLuc, mEPO and eGFP, codon exchange table 2C was used. In this study, for the UG-depleted variants of secNLuc, mEPO and eGFP, codon exchange table 3 was used. In this study, for the C2-depleted variants of secNLuc, mEPO and eGFP, codon exchange table 1A was used. In this study, for the C-depleted variants of secNLuc, mEPO and eGFP, codon exchange table 5 was used. Next, the desired 5′UTR and 3′UTR (detailed in Table 1 in Example 2) were added in silico to obtain the (modified) RNA sequences obtained from the previous step. The 5′UTR was added directly upstream of the (modified) coding sequence, and the 3′UTR was added directly downstream of the (modified) coding sequence. Next, a T7 promoter (sequence TAATACGACTCACTATA (SEQ ID No.1) followed by up to 3 G nucleotides were added in silico to the 5′ end of the sequence. If the selected 5′UTR already had one or more consecutive Guanosine nucleotides at the 5′end, the number of additional Guanosine nucleotides was reduced so that 3 Guanosine nucleotides remain at the 5′end of the 5′UTR and directly downstream of the T7 promoter sequence. Upstream of the obtained sequence, additional nucleotides were added to facilitate accurate de novo DNA synthesis. For this study, 2 nucleotides (GG) were added in silico upstream of the T7 promoter. Downstream of the obtained sequence, additional nucleotides were added to facilitate de novo DNA synthesis. Additional downstream nucleotides were removed in Example 2 by using reverse PCR primers that start exactly at the desired 3′end.
Example 2
[0096] Generation and Purification of mRNAs
[0097] For the wild-type sequence, secreted nano-luciferase DNA was ordered from Promega as a plasmid (pNL3.3). To obtain a linear template, the plasmid was amplified with primers (Fwd primer: tacgtagcgcTAATACGACTCAC (SEQ ID No.2) & Rvs primer: GTATCTTATCATGTCTGCTCGAAG (SEQ ID No.3)) by the Q5 DNA polymerase (annealing temperature 63° C., extension temperature 72° C., annealing time 30 seconds, extension time 20 seconds, 25 cycles of amplification, 2 minute final extension, 10 ng DNA input). Subsequently, the plasmid DNA was digested with DpnI (provide by New England Biolabs (NEB)) for 1 h at 37° C. by adding 20 U of DpnI (1 μl) to the PCR reaction. The digested plasmid DNA and PCR reaction salts and proteins were removed by a Qiagen MinElute PCR cleanup column (Qiagen) according to manufacturer's protocol. The purified DNA template was spectrophotometrically quantified, diluted to 100 ng/μl. mRNA produced from this template was used to validate the luciferase assay.
[0098] For the sequence-engineered mRNAs encoding secreted nanoluciferase and the corresponding wild-type control was DNA encoding the secreted nanoluciferase ordered from and synthesized by IDT (Integrated DNA technologies). The delivered DNA was amplified by PCR with primers (Fwd primer: ggaggTAATACGACTCACTATAGGG (SEQ ID No.4) & Rvs primer: TTTTGTGTTGGTTGTGTTGTGGT (SEQ ID No.5) for the U-depleted and UG-depleted mRNA, or Fwd primer: ggaggTAATACGACTCACTATAGGG (SEQ ID No.4) & Rvs primer: TTTTCTCTTCCTTCTCTTCTCCT (SEQ ID No.6) for the WT, UC-depleted and C-depleted mRNAs) and the Q5 DNA polymerase, according to manufacturer's protocol (annealing temperature 63° C., extension temperature 72° C., annealing time 30 seconds, extension time 20 seconds, 25 cycles of amplification, 2 minute final extension, 10 ng DNA input). PCR reaction salts and proteins were removed by a Qiagen MinElute PCR cleanup column (Qiagen) according to manufacturer's protocol. The purified DNA template was spectrophotometrically quantified, diluted to 100ng/μl.
[0099] 200ng of each of the DNA templates was used as input in a standard T7 RNA polymerase in vitro transcription reaction (according to protocol, NEB HiScribe T7 RNA synthesis kit), including 1 μl of Murine RNAse inhibitor (NEB) per 20 μl of reaction volume to prevent RNAse-mediated degradation of the nascent RNA. The 4 canonical nucleotides (ATP, CTP, UTP, GTP) were used for transcription.
[0100] After 3 h incubation at 37° C., 1 μl of Turbo DNAse (2units, Thermo Fisher Scientific) was added and incubated for 1 h at 37° C. Next, the RNA was A-tailed by E.coli poly(A) polymerase (NEB, according to protocol) to obtain a 150 nt-long polyA-tail. After verification of proper A-tail length, the RNA was purified on RNeasy mini silica columns according to manufacturer's protocol (Qiagen). The purified RNA was twice eluted in 2 times 7 μl of RNase-free MQ and spectrophotometrically quantified. Next, a 5′cap (cap1) was added with vaccinia capping enzyme (NEB) and simultaneous 2′O-methyltransferase (NEB) treatment according to manufacturer's protocol. The completed mRNA was purified on cellulose column (according to Baiersdörfer, M. et al. A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA. Mol. Ther. —Nucleic Acids 15, 26-35 (2019)) to remove dsRNA arising as side-product from the T7 reaction. The eluate was subsequently purified on a Qiagen RNeasy mini column (first step is to add 1470 μl RLT buffer (Qiagen) and 970 μl 100% ETOH (Sigma Aldrich, >99.8%) and add entire mixed volume in steps of 700 μl to the column and elute). Subsequent steps were according to manufacturer's protocol) and the mRNA was eluted in RNAse-free water. The material was spectrophotometrically quantified and diluted to 1 μg/μl with RNase-free MQ.
[0101] All other mRNAs used in this application were synthesized with the method described above, using the 5′UTR and 3′UTR, primers and PCR conditions as shown in Table 1 below.
TABLE-US-00002 TABLE 1 Synthesis details of DNA templates for RNA transcription PCR mRNA 5′UTR 3′UTR Fwd primer Rvs primer conditions Secreted GGGAAACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. nanoluc- (SEQ ID No. 7) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) WT Secreted GGGAAACGCCGCCACC CCACAACACAACCAACACAAAA ggTAATACGACTCACTATAGGG TTTTGTGTTGGTTGTGTTGTGGT Anneal: 63° C. nanoluc- (SEQ ID No. 7) (SEQ ID No. 9) (SEQ ID No. 4) (SEQ ID No. 13) U- depleted Secreted GGGAAACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. nanoluc- (SEQ ID No. 7) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) UC- depleted Secreted GGGAAACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. nanoluc- (SEQ ID No. 7) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) C- depleted Secreted GGGAAACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. nanoluc- (SEQ ID No. 7) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) C2- depleted Secreted GGGAAACGCCGCCACC CCACAACACAACCAACACAAAA gg TAATACGACTCACTATAGGG TTTTGTGTTGGTTGTGTTGTGGT Anneal: 63° C. nanoluc- (SEQ ID No. 7) (SEQ ID No. 9) (SEQ ID No. 4) (SEQ ID No. 13) UG- depleted Murine GGGAAACTGCCAAG GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. EPO- (SEQ ID No. 10) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) WT Murine GGGAAACTGCCAAG CCACAACACAACCAACACAAAA ggTAATACGACTCACTATAGGG TTTTGTGTTGGTTGTGTTGTGGT Anneal: 63° C. EPO- (SEQ ID No. 10) (SEQ ID No. 9) (SEQ ID No. 4) (SEQ ID No. 13) U- depleted Murine GGGAAACTGCCAAG GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. EPO- (SEQ ID No. 10) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) UC- depleted Murine GGGAAACTGCCAAG GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. EPO- (SEQ ID No. 10) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) C- depleted Murine GGGAAACTGCCAAG GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. EPO- (SEQ ID No. 10) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) C2- depleted EGFP- GGGATACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. WT (SEQ ID No. 11) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) EGFP- GGGATACGCCGCCACC CCACAACACAACCAACACAAAA ggTAATACGACTCACTATAGGG TTTTGTGTTGGTTGTGTTGTGGT Anneal: 63° C. U- (SEQ ID No. 11) (SEQ ID No. 9) (SEQ ID No. 4) (SEQ ID No. 13) depleted EGFP- GGGATACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. UC- (SEQ ID No. 11) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) depleted EGFP- GGGATACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. C- (SEQ ID No. 11) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) depleted EGFP GGGATACGCCGCCACC GGAGAAGAGAAGGAAGAGAAAA ggTAATACGACTCACTATAGGG TTTTCTCTTCCTTCTCTTCTCCT Anneal: 63° C. C2- (SEQ ID No. 11) (SEQ ID No. 8) (SEQ ID No. 4) (SEQ ID No. 12) depleted N.B. EPO = Erythropoietin, NanoLuc = nanoluciferase, eGFP = enhanced Green Fluorescent. Protein
Example 3
[0102] Preparation of Lipofectamine MessengerMax-Complexed mRNA
[0103] Because uptake of naked mRNA into the cytosol is minimal to non-existing in the majority of cells, mRNA was complexed with a delivery vehicle (Lipofectamine Messenger-Max) to facilitate uptake in cells in vitro.
[0104] mRNA produced according to Example 2 was complexed with Lipofectamine MessengerMax (ThermoFisherScientific) according to instructions by the manufacturer. Briefly, first mRNA was diluted with sterile Optimem (4° C.) to 20ng/μl and 10 μl of the diluted mRNA was used for a single complexation. 0.3 μl of Lipofectamine MessengerMax reagent was mixed with 10 μl of sterile, pre-warmed (to RT) Optimem medium by pipetting. After 10 minutes of incubation, the entire 10 μl was mixed with 10 μl of pre-diluted mRNA (containing a total of 200 ng of mRNA). After careful mixed by pipetting up and down, the mixed components were incubated for 5 minutes at RT before injection or addition to cell culture.
[0105] For complexing different amounts of mRNA, the volumes of reagents and the final volume scaled proportionally.
Example 4
[0106] Preparation of TransIT-Complexed mRNA
[0107] Some of the mRNA were complexed with another delivery vehicle (TransIT) to facilitate uptake in cells in vivo.
[0108] mRNA produced according to Example 2 was complexed with TransIT (Mirus Bio, Madison, Wis.) according to manufacturer's instructions. Briefly, 1 μg of mRNA (generally 1 μl) was mixed with 98 μl of pre-warmed (to RT) DMEM (Dulbecco's modified Eagle medium), followed by the addition of 1.1 μl of TransIT-mRNA reagent and 0.7 μl of Boost reagent. After combination, the mixture was briefly, gently vortexed and incubated for 2-5 minutes before injection.
[0109] For complexing different amounts of mRNA, the volumes of reagents and the final volume scaled proportionally.
Example 5
[0110] Administration of Formulated mRNAs
[0111] The day before transfection, HeLa cells were plated in 96-well plate at 40% confluency (100 μl of medium (DMEM +10% FCS)/well). 24 h later, HeLa cells, grown to 80% confluency in a 96-well plate were transfected with 10, 50 or 100 ng of secNLuc mRNA complexed with Lipofectamine MessengerMax (Thermo Fisher Scientific) prepared according to Example 3 and incubated for 24 h at 37° C. In case of 100 ng, 10 μl of complexed mRNA solution is mixed with 100 μl of medium and added to the cells. In case of 50 ng, 5 μl of complexed mRNA solution is mixed with 5 μl of Optimem medium and then subsequently mixed with 100 μl of medium and added to cells. In case of 10 ng, 1 μl of complexed mRNA solution is mixed with 9 μl of Optimem medium and then subsequently mixed with 100 μl of medium and added to cells.
[0112] After incubation, the entire medium volume was removed and a sample was taken for analysis Animal studies were performed in accordance with the Dutch animal welfare regulations and approved by the Central Animal Experiments Committee (VD103002015270). 1 μg of wild-type (WT), U-, UC- or UG-depleted mEpo mRNA was formulated with TransIT (MirusBio) according to Example 4. After mixing, the formulation was incubated for 5 minutes at RT and directly injected into mice. For this, 10-12 week-old female BALB/cJRj mice (Janvier Labs) were intraperitoneally injected with 100 μl of respective mRNA formulation. After 6 and 24 hours, blood was collected via the tail vein in a Heparin-coated capillary tube. Heparin-plasma was transferred to a 1.5-ml Eppendorf tube and stored at −20° C. until further use. Plasma samples were tested for mEpo using the mEpo assay (R&D) as described above using 5-fold dilution of the plasma samples in Calibrator Diluent.
Example 6
Detection of Protein Expression
[0113] For measuring secreted nanoluciferase, medium was collected 24 hours after transfection. Luciferase activity was detected with the Nano-Glo Luciferase Assay System (Promega) according to manufacturer's specifications. Importantly, the assay buffer was thawed and equilibrated to RT for more than 1 hour at RT.
[0114] For measuring eGFP, medium was removed 24 h after transfection and cells were washed twice with PBS and cell lysates were prepared by adding 30 μl lysis buffer (10 mM TrisHCl pH7+10% glycerol, 2% Tween, 2% Triton X-100 and 0.31 mg/ml freshly added DTT) per well. Cell were incubated for 20 minutes at 37° C. and cell lysates were collected and pooled from 3 wells. Fluorescence was measured using a 485/20 excitation and 528/20 emission filter on a plate reader.
[0115] mEpo concentrations were measured in supernatant collected 24 hours after transfection, using the mEpo assay (R&D Systems) according to the manufacturer's protocol. In short, 50 μl Assay Diluent was added to pre-coated wells and supplemented with 50 μl prepared standard or supernatants diluted in Calibrator Diluent. Wells were incubated for 2 hours at RT with shaking. Wells were washed 5 times with 200 μl wash buffer and 100 μl Mouse Epo conjugate was added to each well. After incubating for 2 hours at RT with shaking, wells were washed 5 times with 200 μl wash buffer. Wells were developed with 100 μl Substrate Solution per well for 20-30 minutes at RT in the dark, depending on the strength of the signal. The reaction was stopped by adding 100 μl Stop Solution to each well and the signal was measured at 450 nm in a plate reader (Biorad).
[0116] MCP-1 was measured in supernatants that were collected after 24 hours, using the mouse MCP-1 ELISA (R&D Systems) according to the manufacturer's protocols. Shortly, a Costar Maxisorb 96-well plate was coated overnight at 4° C. with 100 μl/well Capture Antibody. Wells washed 3× with 250-300 μl/well Wash Buffer (0.05% Tween-20 in PBS) and blocked for 1 hour at RT with 250 μl 1% (98%-pure) BSA in PBS. Subsequently, wells were washed 3× with 250-300 μl Wash Buffer. Pre-diluted samples and recombinant MCP-1 standard was transferred to the wells and incubated for 2 hours at RT. Wells were again washed 5× with 250-300 μl Wash Buffer and incubated for 1 hour at RT with 100 μl/well Detection Antibody. After washing the wells washed 5× with 250-300 μl Wash Buffer, wells were incubated for 30 minutes at RT with 100 μl/well Avidin-HRP, and washed as described above. 100 μl/well TMB Solution was added and incubated for 10-15 minutes. The reaction was stopped by adding 50 μl/well 2 M H2SO4 and measured at 450 nm using a plate reader (Biorad).
RESULTS OF DEPLETION EXPERIMENTS
[0117] As can be seen in
[0118] Interestingly, the combination of Uridine depletion with Cytidine depletion resulted in even higher protein expression, suggesting an additive effect of Cytidine nucleotides on activation of innate immune receptors.
[0119] Surprisingly, combination of Uridine depletion with Guanosine depletion, typically creating an mRNA rich in Cytidine, resulted in a decreased protein expression compared to wild-type. This result is surprising because Uridine and Guanosine are able to bind each other in addition to their preferred binding partners Adenosine and Cytidine, respectively. Reduction of both Uridine and Guanosine would have been expected to reduce innate immunity and thus boost protein expression by reducing the options for extended dsRNA formation in an RNA structure. In addition, several studies (e.g. Zhang, Z. et al. Structural Analysis Reveals that Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA Immunity 45, 737-748 (2016) and Tanji, H. et al. Toll-like receptor 8 senses degradation products of single-stranded RNA. Nat. Struct. Mol. Biol. (2015). doi:10.1038/nsmb.2943) have indicated the Uridine in the presence of Guanosine would be particularly activating for TLR7 and TLR8, two of the innate immune sensors.
[0120] In a further experiment, clarification on the role of Cytidine in the reduction of mRNA mediated protein expression was obtained Similar to the previous experiment, UC-depleted mRNA shows a higher expression than U-depleted mRNA. Interestingly, C-depletion by itself also resulted in increased secreted nanoluciferase expression compared to U-depleted and WT mRNA. Further strengthening the case for Cytidine involvement is the dose-response effect that was obtained by further reducing the number of Cytidine nucleotides in C2-depleted mRNA compared to C-depleted mRNA, resulting in even higher protein expression. This effect was maintained across all doses tested.
[0121] Similar results were obtained with murine EPO coding mRNA, both Uridine and Cytidine depletion, alone or in combination, resulted in enhanced protein expression in HeLa cells. Intra-peritoneal injection of the mRNAs in mice resulted in significantly increased circulating mEPO plasma levels at 6h after injection for the U-depleted and UC-depleted mRNAs. The differences with wild-type and UG-depleted mRNAs were even greater, suggesting the role of innate immune activation reduction in protein expression from mRNA is greater in vivo than in HeLa cells. Furthermore, it strengthens the case for Cytidine-depletion or UC-depletion mediated de-immunization of mRNAs to be used for therapeutic purposes.
[0122] Finally, using eGFP, a similar protein expression effect was observed for depleted mRNAs coding for an intracellular protein. In order of increasing protein expression: WT, U-depleted, UC-depleted, C-depleted and C2-depleted. Interestingly, again the highest protein expression was obtained with C-depleted and C2-depleted mRNAs. The observed effects were maintained over all doses.
CODING SEQUENCES
[0123]
TABLE-US-00003 Coding sequence 1-WT coding sequence secreted NanoLuc (SEQ ID No: 14) ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCCT GGTGTTGCCTGCTGCCTTCCCTGCCCCAGTCTTCACACTCGAAGATTTCGTTGGGGACT GGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCA GTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGG TGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATC ACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACAT GATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATC ACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAAC CCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGT GCGAACGCATTCTGGCGTAA Coding sequence 2-WT coding sequence murine Erythropoietin (SEQ ID No: 15) ATGGGGGTGCCCGAACGTCCCACCCTGCTGCTTTTACTCTCCTTGCTACTGATTCCTCTG GGCCTCCCAGTCCTCTGTGCTCCCCCACGCCTCATCTGCGACAGTCGAGTTCTGGAGAGG TACATCTTAGAGGCCAAGGAGGCAGAAAATGTCACGATGGGTTGTGCAGAAGGTCCCAG ACTGAGTGAAAATATTACAGTCCCAGATACCAAAGTCAACTTCTATGCTTGGAAAAGAAT GGAGGTGGAAGAACAGGCCATAGAAGTTTGGCAAGGCCTGTCCCTGCTCTCAGAAGCCA TCCTGCAGGCCCAGGCCCTGCTAGCCAATTCCTCCCAGCCACCAGAGACCCTTCAGCTTC ATATAGACAAAGCCATCAGTGGTCTACGTAGCCTCACTTCACTGCTTCGGGTACTGGGA GCTCAGAAGGAATTGATGTCGCCTCCAGATACCACCCCACCTGCTCCACTCCGAACACTC ACAGTGGATACTTTCTGCAAGCTCTTCCGGGTCTACGCCAACTTCCTCCGGGGGAAACTG AAGCTGTACACGGGAGAGGTCTGCAGGAGAGGGGACAGGTGA Coding sequence 3-WT coding sequence eGFP (SEQ ID No: 16) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCAC CTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC CCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACA TGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACC ATCTCCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA CACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCC TGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAG CAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGC CCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA GCTGTACAAGTAA
PROTEIN SEQUENCES
[0124] All used/sequence-engineered murine EPO (Mus musculus) nucleic acid sequences encode the same murine EPO protein with the following amino acid sequence(SEQ ID No:17) :
TABLE-US-00004 MGVPERPTLLLLLSLLLIPL GLPVLCAPPRLICDSRVLER YILEAKEAENVTMGCAEGPR LSENITVPDTKVNFYAWKRM EVEEQAIEVWQGLSLLSEAI LQAQALLANSSQPPETLQLH IDKAISGLRSLTSLLRVLGA QKELMSPPDTTPPAPLRTLT VDTFCKLFRVYANFLRGKLK LYTGEVCRRGDR*
All used/sequence-engineered eGFP (extensively mutated from Aequorea victoria) nucleic acid sequences encode the same eGFP protein with the following amino acid sequence (SEQ ID No:18):
TABLE-US-00005 MVSKGEELFTGVVPILVELD GDVNGHKFSVSGEGEGDATY GKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPDHMK QHDFFKSAMPEGYVQERTIS FKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKN GIKANFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNH YLSTQSALSKDPNEKRDHMV LLEFVTAAGITLGMDELYK*
All used/sequence-engineered secreted nanoluciferase (developed by Promega) nucleic acid sequences encode the same nanoluciferase protein with the following amino acid sequence (SEQ ID No:19):
TABLE-US-00006 MNSFSTSAFGPVAFSLGLLL VLPAAFPAPVFTLEDFVGDW RQTAGYNLDQVLEQGGVSSL FQNLGVSVTPIQRIVLSGEN GLKIDIHVIIPYEGLSGDQM GQIEKIFKVVYPVDDHHFKV ILHYGTLVIDGVTPNMIDYF GRPYEGIAVFDGKKITVTGT LWNGNKIIDERLINPDGSLL FRVTINGVTGWRLCERILA*
NUCLEIC ACID SEQUENCES
[0125]
TABLE-US-00007 Secreted NanoLuc-WT (assay control) (SEQ ID No: 20) GGGATACGCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCT CCCTGGGCCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTCTTCACACTCGAA GATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAAC AGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAG GATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTAT GAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTAC CCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACG GGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTT CGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGA CGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTG ACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAAGGCCGCGACTCTAGAGTCGGG GCGGCCGGCCGCTTCGAGCAGACATGATAAGATAC Secreted NanoLuc-WT (control to other mRNAs) (SEQ ID No: 21) GGGAAACGCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTC TCCCTGGGCCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTCTTCACACTCGA AGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAA CAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAA GGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTA TGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTA CCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGAC GGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGT TCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCG ACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGT GACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAAGGAGAAGAGAAGGAAGAGAA AA Secreted NanoLuc-U-depleted (maximum exchange) (SEQ ID No: 22) GGGAAACGCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGACCAGTCGCCTTC TCCCTGGGCCTGCTCCTGGTGCTCCCCGCAGCCTTCCCCGCCCCAGTCTTCACACTCGA AGACTTCGTCGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTCGA ACAGGGAGGAGTGTCCAGCCTCTTCCAGAACCTCGGGGTGTCCGTAACTCCGATCCAA AGGATCGTCCTGAGCGGAGAAAACGGGCTGAAGATCGACATCCACGTCATCATCCCG TACGAAGGACTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATCTTCAAGGTGGTG TACCCCGTGGACGACCACCACTTCAAGGTGATCCTGCACTACGGCACACTGGTAATCG ACGGGGTCACGCCGAACATGATCGACTACTTCGGACGGCCGTACGAAGGCATCGCCG TGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATCA TCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGG AGTGACCGGCTGGCGGCTGTGCGAACGCATCCTGGCGTAACCACAACACAACCAACA CAAAA Secreted NanoLuc-UC-depleted (maximum exchange) (SEQ ID No: 23) GGGAAACGCCGCCACCATGAACAGCTTCAGCACAAGCGCATTCGGACCAGTGGCATT CAGCCTGGGACTGCTGCTGGTGCTGCCAGCAGCATTCCCAGCACCAGTCTTCACACTG GAGGACTTCGTGGGGGACTGGAGACAGACAGCAGGATACAACCTGGACCAGGTCCTG GAGCAGGGAGGAGTGAGCAGCCTGTTCCAGAACCTGGGGGTGAGCGTGACACCAATC CAGAGAATCGTCCTGAGCGGAGAGAACGGGCTGAAGATCGACATCCACGTCATCATC CCATACGAGGGACTGAGCGGAGACCAGATGGGACAGATCGAGAAGATCTTCAAGGTG GTGTACCCAGTGGACGACCACCACTTCAAGGTGATCCTGCACTACGGAACACTGGTGA TCGACGGGGTGACACCAAACATGATCGACTACTTCGGAAGACCATACGAGGGAATCG CAGTGTTCGACGGAAAGAAGATCACAGTGACAGGGACACTGTGGAACGGAAACAAGA TCATCGACGAGAGACTGATCAACCCAGACGGAAGCCTGCTGTTCAGAGTGACAATCA ACGGAGTGACAGGATGGAGACTGTGCGAGAGAATCCTGGCATAAGGAGAAGAGAAG GAAGAGAAAA Secreted NanoLuc-UG-depleted (maximum exchange) (SEQ ID No: 24) GGGAAACGCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGACCAGTCGCCTTC TCCCTCGGCCTCCTCCTCGTCCTCCCAGCAGCCTTCCCAGCCCCAGTCTTCACACTCGA AGACTTCGTCGGAGACTGGCGACAAACAGCCGGCTACAACCTCGACCAAGTCCTCGA ACAAGGAGGAGTCTCCAGCCTCTTCCAAAACCTCGGAGTCTCCGTAACACCAATCCAA AGAATCGTCCTCAGCGGAGAAAACGGACTCAAAATCGACATCCACGTCATCATCCCAT ACGAAGGACTCAGCGGCGACCAAATGGGCCAAATCGAAAAAATCTTCAAAGTCGTCT ACCCAGTCGACGACCACCACTTCAAAGTCATCCTCCACTACGGCACACTCGTAATCGA CGGAGTCACACCAAACATGATCGACTACTTCGGACGCCCATACGAAGGCATCGCCGTC TTCGACGGCAAAAAAATCACAGTAACAGGAACCCTCTGGAACGGCAACAAAATCATC GACGAGCGCCTCATCAACCCCGACGGCTCCCTCCTCTTCCGAGTAACCATCAACGGAG TCACCGGCTGGCGCCTCTGCGAACGCATCCTCGCATAACCACAACACAACCAACACAA AA Secreted NanoLuc-C-depleted (maximum exchange of only C-containing but not U-containing codons) (SEQ ID No: 25) GGGAAACGCCGCCACCATGAACTCCTTCTCCACAAGCGCATTCGGTCCAGTTGCATTC TCCCTGGGACTGCTCCTGGTGTTGCCTGCTGCATTCCCTGCACCAGTCTTCACACTCGA AGATTTCGTTGGGGACTGGCGACAGACAGCAGGATACAACCTGGACCAAGTCCTTGA ACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAA AGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGT ATGAAGGTCTGAGCGGAGACCAAATGGGACAGATCGAAAAAATTTTTAAGGTGGTGT ACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGAACACTGGTAATCGA CGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGAATCGCAGTG TTCGACGGAAAAAAGATCACTGTAACAGGGACACTGTGGAACGGAAACAAAATTATC GACGAGCGGCTGATCAACCCAGACGGATCCCTGCTGTTCCGAGTAACAATCAACGGA GTGACAGGATGGCGGCTGTGCGAACGGATTCTGGCGTAAGGAGAAGAGAAGGAAGA GAAAA Secreted NanoLuc-C2-depleted (maximum exchange of all C-containing codons) (SEQ ID No: 26) GGGAAACGCCGCCACCATGAACAGTTTCAGTACAAGCGCATTCGGTCCAGTTGCATTC AGTCTGGGACTGCTGCTGGTGTTGCCTGCTGCATTCCCTGCACCAGTGTTCACACTGGA AGATTTCGTTGGGGACTGGCGACAGACAGCAGGATACAACCTGGACCAAGTGCTTGA ACAGGGAGGTGTGAGTAGTTTGTTTCAGAATCTGGGGGTGAGTGTAACTCCGATACAA AGGATTGTGCTGAGCGGTGAAAATGGGCTGAAGATAGACATACATGTGATAATACCG TATGAAGGTCTGAGCGGAGACCAAATGGGACAGATAGAAAAAATTTTTAAGGTGGTG TACCCTGTGGATGATCATCACTTTAAGGTGATACTGCACTATGGAACACTGGTAATAG ACGGGGTTACGCCGAACATGATAGACTATTTCGGACGGCCGTATGAAGGAATAGCAG TGTTCGACGGAAAAAAGATAACTGTAACAGGGACACTGTGGAACGGAAACAAAATTA TAGACGAGCGGCTGATAAACCCAGACGGAAGTCTGCTGTTCCGAGTAACAATAAACG GAGTGACAGGATGGCGGCTGTGCGAACGGATTCTGGCGTAAGGAGAAGAGAAGGAA GAGAAAA eGFP-WT (SEQ ID No: 27) GGGATACGCCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCC CATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGC TTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCG AAGGCTACGTCCAGGAGCGCACCATCTCCTTCAAGGACGACGGCAACTACAAGACCC GCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACA GCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGGAGAAGAG AAGGAAGAGAAAA eGFP-U-depleted (maximum exchange) (SEQ ID No: 28) GGGATACGCCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCC CATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA GGGCGAGGGCGACGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGC TTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCG AAGGCTACGTCCAGGAGCGCACCATCTCCTTCAAGGACGACGGCAACTACAAGACCC GCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACA GCCACAACGTCTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGACCACATGGTCCTGCTGGAGTT CGTGACCGCCGCCGGGATCACACTCGGCATGGACGAGCTGTACAAGTAACCACAACA CAACCAACACAAAA eGFP-UC-depleted (maximum exchange) (SEQ ID No: 29) GGGATACGCCGCCACCATGGTGAGCAAGGGGGAGGAGCTGTTCACAGGGGTGGTGCC AATCCTGGTCGAGCTGGACGGGGACGTAAACGGGCACAAGTTCAGCGTGAGCGGGGA GGGGGAGGGGGACGCAACATACGGGAAGCTGACACTGAAGTTCATCTGCACAACAGG GAAGCTGCCAGTGCCATGGCCAACACTCGTGACAACACTGACATACGGGGTGCAGTG CTTCAGCAGATACCCAGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCAATGCCA GAAGGGTACGTCCAGGAGAGAACAATCAGCTTCAAGGACGACGGGAACTACAAGACA AGAGCAGAGGTGAAGTTCGAGGGGGACACACTGGTGAACAGAATCGAGCTGAAGGG GATCGACTTCAAGGAGGACGGGAACATCCTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTCTACATCATGGCAGACAAGCAGAAGAACGGGATCAAGGCAAACTT CAAGATCAGACACAACATCGAGGACGGGAGCGTGCAGCTCGCAGACCACTACCAGCA GAACACACCAATCGGGGACGGGCCAGTGCTGCTGCCAGACAACCACTACCTGAGCAC ACAGAGCGCACTGAGCAAAGACCCAAACGAGAAGAGAGACCACATGGTCCTGCTGGA GTTCGTGACAGCAGCAGGGATCACTCTCGGGATGGACGAGCTGTACAAGTAAGGAGA AGAGAAGGAAGAGAAAA eGFP-UG-depleted (maximum exchange) (SEQ ID No: 30) GGGATACGCCGCCACCATGGTCAGCAAAGGCGAAGAACTCTTCACCGGAGTCGTCCC CATCCTCGTCGAACTCGACGGCGACGTAAACGGCCACAAATTCAGCGTCTCCGGCGAA GGCGAAGGCGACGCCACCTACGGCAAACTCACCCTCAAATTCATCTGCACCACCGGCA AACTCCCCGTCCCCTGGCCCACCCTCGTCACCACCCTCACCTACGGCGTCCAATGCTTC AGCCGCTACCCCGACCACATGAAACAACACGACTTCTTCAAATCCGCCATGCCCGAAG GCTACGTCCAAGAACGCACCATCTCCTTCAAAGACGACGGCAACTACAAAACCCGCG CCGAAGTCAAATTCGAAGGCGACACCCTCGTCAACCGCATCGAACTCAAAGGCATCG ACTTCAAAGAAGACGGCAACATCCTCGGACACAAACTCGAATACAACTACAACAGCC ACAACGTCTACATCATGGCCGACAAACAAAAAAACGGCATCAAAGCCAACTTCAAAA TCCGCCACAACATCGAAGACGGCAGCGTCCAACTCGCCGACCACTACCAACAAAACA CCCCCATCGGCGACGGCCCCGTCCTCCTCCCCGACAACCACTACCTCAGCACCCAATC CGCCCTCAGCAAAGACCCCAACGAAAAACGCGACCACATGGTCCTCCTCGAATTCGTC ACCGCCGCCGGAATCACACTCGGCATGGACGAACTCTACAAATAACCACAACACAAC CAACACAAAA eGFP- C-depleted (maximum exchange of only C-containing but not U- containing codons) (SEQ ID No: 31) GGGATACGCCGCCACCATGGTGAGCAAGGGAGAGGAGCTGTTCACAGGGGTGGTGCC AATCCTGGTCGAGCTGGACGGAGACGTAAACGGACACAAGTTCAGCGTGTCCGGAGA GGGAGAGGGAGATGCAACATACGGAAAGCTGACACTGAAGTTCATCTGCACAACAGG AAAGCTGCCAGTGCCATGGCCAACACTCGTGACAACACTGACATACGGAGTGCAGTG CTTCAGCCGGTACCCAGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCAATGCCA GAAGGATACGTCCAGGAGCGGACAATCTCCTTCAAGGACGACGGAAACTACAAGACA CGGGCAGAGGTGAAGTTCGAGGGAGACACACTGGTGAACCGGATCGAGCTGAAGGGA ATCGACTTCAAGGAGGACGGAAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC AGCCACAACGTCTATATCATGGCAGACAAGCAGAAGAACGGAATCAAGGCAAACTTC AAGATCCGGCACAACATCGAGGACGGAAGCGTGCAGCTCGCAGACCACTACCAGCAG AACACACCAATCGGAGACGGACCAGTGCTGCTGCCAGACAACCACTACCTGAGCACA CAGTCCGCACTGAGCAAAGACCCAAACGAGAAGCGGGATCACATGGTCCTGCTGGAG TTCGTGACAGCAGCAGGGATCACTCTCGGAATGGACGAGCTGTACAAGTAAGGAGAA GAGAAGGAAGAGAAAA eGFP-C2-depleted (maximum exchange of all C-containing codons) (SEQ ID No: 32) GGGATACGCCGCCACCATGGTGAGCAAGGGAGAGGAGCTGTTCACAGGGGTGGTGCC AATACTGGTGGAGCTGGACGGAGACGTAAACGGACACAAGTTCAGCGTGAGTGGAGA GGGAGAGGGAGATGCAACATACGGAAAGCTGACACTGAAGTTCATATGCACCACAGG AAAGCTGCCAGTGCCATGGCCAACACTGGTGACAACACTGACATACGGAGTGCAGTG CTTCAGCCGGTACCCAGACCACATGAAGCAGCACGACTTCTTCAAGAGTGCAATGCCA GAAGGATACGTGCAGGAGCGGACAATAAGTTTCAAGGACGACGGAAACTACAAGACA CGGGCAGAGGTGAAGTTCGAGGGAGACACACTGGTGAACCGGATAGAGCTGAAGGG AATAGACTTCAAGGAGGACGGAAACATACTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTGTATATAATGGCAGACAAGCAGAAGAACGGAATAAAGGCAAACTT CAAGATACGGCACAACATAGAGGACGGAAGCGTGCAGCTGGCAGACCACTACCAGCA GAACACACCAATAGGAGACGGACCAGTGCTGCTGCCAGACAACCACTACCTGAGCAC ACAGAGTGCACTGAGCAAAGACCCAAACGAGAAGCGGGATCACATGGTGCTGCTGGA GTTCGTGACAGCAGCAGGGATAACTCTGGGAATGGACGAGCTGTACAAGTAAGGAGA AGAGAAGGAAGAGAAAA mEPO-WT (SEQ ID No: 33) GGGAAACTGCCAAGATGGGGGTGCCCGAACGTCCCACCCTGCTGCTTTTACTCTCCTT GCTACTGATTCCTCTGGGCCTCCCAGTCCTCTGTGCTCCCCCACGCCTCATCTGCGACA GTCGAGTTCTGGAGAGGTACATCTTAGAGGCCAAGGAGGCAGAAAATGTCACGATGG GTTGTGCAGAAGGTCCCAGACTGAGTGAAAATATTACAGTCCCAGATACCAAAGTCA ACTTCTATGCTTGGAAAAGAATGGAGGTGGAAGAACAGGCCATAGAAGTTTGGCAAG GCCTGTCCCTGCTCTCAGAAGCCATCCTGCAGGCCCAGGCCCTGCTAGCCAATTCCTCC CAGCCACCAGAGACCCTTCAGCTTCATATAGACAAAGCCATCAGTGGTCTACGTAGCC TCACTTCACTGCTTCGGGTACTGGGAGCTCAGAAGGAATTGATGTCGCCTCCAGATAC CACCCCACCTGCTCCACTCCGAACACTCACAGTGGATACTTTCTGCAAGCTCTTCCGGG TCTACGCCAACTTCCTCCGGGGGAAACTGAAGCTGTACACGGGAGAGGTCTGCAGGA GAGGGGACAGGTGAGGAGAAGAGAAGGAAGAGAAAA mEPO-U-depleted (maximum exchange) (SEQ ID No: 34) GGGAAACTGCCAAGATGGGGGTGCCCGAACGACCCACCCTGCTGCTCTTACTCTCCCT CCTACTGATCCCCCTGGGCCTCCCAGTCCTCTGCGCACCCCCACGCCTCATCTGCGACA GCCGAGTCCTGGAGAGGTACATCTTAGAGGCCAAGGAGGCAGAAAACGTCACGATGG GATGCGCAGAAGGACCCAGACTGAGCGAAAACATCACAGTCCCAGACACCAAAGTCA ACTTCTACGCATGGAAAAGAATGGAGGTGGAAGAACAGGCCATAGAAGTCTGGCAAG GCCTGTCCCTGCTCTCAGAAGCCATCCTGCAGGCCCAGGCCCTGCTAGCCAACTCCTC CCAGCCACCAGAGACCCTCCAGCTCCACATAGACAAAGCCATCAGCGGACTACGAAG CCTCACATCACTGCTCCGGGTACTGGGAGCACAGAAGGAACTCATGTCGCCCCCAGAC ACCACCCCACCCGCACCACTCCGAACACTCACAGTGGACACATTCTGCAAGCTCTTCC GGGTCTACGCCAACTTCCTCCGGGGGAAACTGAAGCTGTACACGGGAGAGGTCTGCA GGAGAGGGGACAGGTAACCACAACACAACCAACACAAAA mEPO-UC-depleted (maximum exchange) (SEQ ID No: 35) GGGAAACTGCCAAGATGGGGGTGCCAGAACGACCAACACTGCTGCTCCTACTCAGCTT GCTACTGATCCCACTGGGGCTCCCAGTCCTCTGCGCACCACCAAGACTCATCTGCGAC AGCCGAGTACTGGAGAGGTACATCCTAGAGGCAAAGGAGGCAGAAAACGTCACGATG GGATGCGCAGAAGGACCAAGACTGAGCGAAAACATCACAGTCCCAGACACAAAAGTC AACTTCTACGCATGGAAAAGAATGGAGGTGGAAGAACAGGCAATAGAAGTATGGCAA GGGCTGAGCCTGCTCAGCGAAGCAATCCTGCAGGCACAGGCACTGCTAGCAAACAGC AGCCAGCCACCAGAGACACTCCAGCTCCACATAGACAAAGCAATCAGCGGACTACGA AGCCTCACTAGCCTGCTCAGGGTACTGGGAGCACAGAAGGAATTGATGTCGCCACCA GACACAACACCACCAGCACCACTCCGAACACTCACAGTGGACACTTTCTGCAAGCTCT TCAGGGTCTACGCAAACTTCCTCAGGGGGAAACTGAAGCTGTACACGGGAGAGGTCT GCAGGAGAGGGGACAGGTGAGGAGAAGAGAAGGAAGAGAAAA mEPO-UG-depleted (maximum exchange) (SEQ ID No: 36) GGGAAACTGCCAAGATGGGAGTCCCCGAACGACCCACCCTCCTCCTCTTACTCTCCCT CCTACTCATCCCACTCGGCCTCCCAGTCCTCTGCGCACCCCCACGCCTCATCTGCGACA GCCGAGTCCTCGAAAGATACATCTTAGAAGCCAAAGAAGCAGAAAACGTCACAATGG GATGCGCAGAAGGACCCAGACTCAGCGAAAACATCACAGTCCCAGACACCAAAGTCA ACTTCTACGCATGGAAAAGAATGGAAGTCGAAGAACAAGCCATAGAAGTCTGGCAAG GCCTCTCCCTCCTCTCAGAAGCCATCCTCCAAGCCCAAGCCCTCCTAGCCAACTCCTCC CAACCACCAGAAACCCTCCAACTCCACATAGACAAAGCCATCAGCGGACTACGAAGC CTCACATCACTCCTCCGCGTACTCGGAGCACAAAAAGAACTCATGTCACCACCAGACA CCACCCCACCAGCACCACTCCGAACACTCACAGTCGACACATTCTGCAAACTCTTCCG CGTCTACGCCAACTTCCTCCGCGGAAAACTCAAACTCTACACAGGAGAAGTCTGCAGA AGAGGAGACAGATAACCACAACACAACCAACACAAAA mEPO-C-depleted (maximum exchange of only C-containing but not U- containing codons) (SEQ ID No: 37) GGGAAACTGCCAAGATGGGGGTGCCAGAACGTCCAACACTGCTGCTTTTACTCTCCTT GCTACTGATTCCTCTGGGACTCCCAGTCCTCTGTGCTCCACCACGGCTCATCTGCGACA GTCGAGTTCTGGAGAGGTACATCTTAGAGGCAAAGGAGGCAGAAAATGTCACGATGG GTTGTGCAGAAGGTCCAAGACTGAGTGAAAATATTACAGTCCCAGATACAAAAGTCA ACTTCTATGCTTGGAAAAGAATGGAGGTGGAAGAACAGGCAATAGAAGTTTGGCAAG GACTGTCCCTGCTCTCAGAAGCAATCCTGCAGGCACAGGCACTGCTAGCAAATTCCTC CCAGCCACCAGAGACACTTCAGCTTCATATAGACAAAGCAATCAGTGGTCTACGTAGC CTCACTTCACTGCTTCGGGTACTGGGAGCTCAGAAGGAATTGATGTCGCCTCCAGATA CAACACCACCTGCTCCACTCCGAACACTCACAGTGGATACTTTCTGCAAGCTCTTCCG GGTCTACGCAAACTTCCTCCGGGGGAAACTGAAGCTGTACACGGGAGAGGTCTGCAG GAGAGGGGACAGGTGAGGAGAAGAGAAGGAAGAGAAAA mEPO-C2-depleted (maximum exchange of all C-containing codons) (SEQ ID No: 38) GGGAAACTGCCAAGATGGGGGTGCCAGAACGTCCAACACTGCTGCTTTTACTGAGTTT GCTACTGATTCCTCTGGGACTGCCAGTGCTGTGTGCTCCACCACGGCTGATATGCGAC AGTCGAGTTCTGGAGAGGTACATATTAGAGGCAAAGGAGGCAGAAAATGTGACGATG GGTTGTGCAGAAGGTCCAAGACTGAGTGAAAATATTACAGTGCCAGATACAAAAGTG AACTTCTATGCTTGGAAAAGAATGGAGGTGGAAGAACAGGCAATAGAAGTTTGGCAA GGACTGAGTCTGCTGAGTGAAGCAATACTGCAGGCACAGGCACTGCTAGCAAATAGT AGTCAGCCACCAGAGACACTTCAGCTTCATATAGACAAAGCAATAAGTGGTCTACGTA GCCTGACTAGTCTGCTTCGGGTACTGGGAGCTCAGAAGGAATTGATGAGTCCTCCAGA TACAACACCACCTGCTCCACTGCGAACACTGACAGTGGATACTTTCTGCAAGCTGTTC CGGGTGTACGCAAACTTCCTGCGGGGGAAACTGAAGCTGTACACGGGAGAGGTGTGC AGGAGAGGGGACAGGTGAGGAGAAGAGAAGGAAGAGAAAA
CODON EXCHANGE TABLES
[0126] The following codon exchange tables are used by the algorithm to generate a new coding sequence for the messenger RNA with the desired base-usage. The tables are to be used as examples only; any combination might be used that leads to the general effect of reducing the Cytidine or Uridine and Cytidine content of the messenger RNA.
Cytidine-depletion without the intention to reduce Uridine, although this might happen to a minor extent. Also, the exchange aims to respect codon usage frequency, by exchanging high frequency codons with high frequency codons, and exchange low frequency codons with low frequency codons.
TABLE-US-00008 Codon exchange table 1A Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 CTT TTG uni 13.2 12.9 L 2 CTC CTG uni 19.6 39.6 L 3 ATC ATA uni 20.8 7.5 I* 4 GTC GTG uni 14.5 28.1 V* 5 TCT AGT uni 15.2 12.1 S 6 TCC AGT uni 17.7 12.1 S 7 TCA AGT uni 12.2 12.1 S 8 TCG AGT uni 4.4 12.1 S* 9 CCC CCA uni 19.8 16.9 P 10 ACC ACA uni 18.9 15.1 T 11 GCC GCA uni 27.7 15.8 A* 12 CGC CGG uni 10.4 11.4 R 13 GGC GGA uni 22.2 16.5 G *Reduced frequency Note: Rules are to change every C to A or G or U. Reducing C takes precedent on codon frequency.
Cytidine-depletion without the intention to reduce Uridine, although this might happen to a minor extent. The reduction of cytidine is combined with exchanging the codon for the highest frequency codon available. This is also applied to codons that would not be changed because of Cytidine content. Cytidine reduction takes precedent over codon frequency optimization.
TABLE-US-00009 Codon exchange table 1B Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTA TTG uni 7.7 12.9 L 2 CTT CTG uni 13.2 39.6 L 3 CTC CTG uni 19.6 39.6 L 4 CTA CTG uni 7.2 39.6 L 5 ATC ATA uni 20.8 7.5 I 6 GTT GTG uni 11 28.1 V 7 GTC GTG uni 14.5 28.1 V 8 GTA GTG uni 7.1 28.1 V 9 TCT AGC uni 15.2 19.5 S 10 TCC AGC uni 17.7 19.5 S 11 TCA AGC uni 12.2 19.5 S 12 TCG AGC uni 4.4 19.5 S 13 CCC CCA uni 19.8 16.9 P 14 CCG CCA uni 6.9 16.9 P 15 ACT ACA uni 13.1 15.1 T 16 ACC ACA uni 18.1 15.1 T 17 ACG ACA uni 6.1 15.1 T 18 CAA CAG uni 12.3 34.2 Q 19 AAA AAG uni 24.4 31.9 K 20 GAA GAG uni 29 39.6 E 21 CGT AGA uni 4.5 12.2 R 22 CGC AGA uni 10.4 12.2 R 23 CGA AGA uni 6.2 12.2 R 24 CGG AGA uni 11.4 12.2 R 25 AGG AGA uni 12 12.2 R 26 GGT GGA uni 10.8 16.5 G 27 GGC GGA uni 22.2 16.5 G 28 GGG GGA uni 16.5 16.5 G Note: Rules are: change every C to A or G but not U. Reducing C takes precedent on codon frequency. If high codon frequency requires introduction of C or U, then take another lower codon frequency or don't change.
Cytidine-depletion combined with Uridine-depletion, with Cytidine taking precedent over Uridine. Also, the exchange aims to respect codon usage frequency, by exchanging high frequency codons with high frequency codons, and exchange low frequency codons with low frequency codons.
TABLE-US-00010 Codon exchange table 2A Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTC TTT uni 20.3 17.6 F 2 CTA TTA uni 7.2 7.7 L 3 CTT TTG uni 13.2 12.9 L 4 ATT ATA uni 16 7.5 I 5 ATC ATA uni 20.8 7.5 I 6 GTT GTA uni 11 7.1 V 7 GTC GTG uni 14.5 28.1 V 8 TCT AGC uni 15.2 19.5 S 9 TCC AGC uni 17.7 19.5 S 10 TCA AGC uni 12.2 19.5 S 11 CCT CCA uni 17.5 16.9 P 12 CCC CCA uni 19.8 16.9 P 13 ACT ACA uni 13.1 15.1 T 14 ACC ACA uni 18.9 15.1 T 15 GCT GCA uni 18.4 15.8 A 16 GCC GCA uni 27.7 15.8 A 17 TAC TAT uni 15.3 12.2 Y 18 CAC CAT uni 15.1 10.9 H 19 AAC AAT uni 19.1 17 N 20 GAC GAT uni 25.1 21.8 D 21 TGC TGT uni 12.6 10.6 C 22 CGT CGA uni 4.5 6.2 R 23 CGC AGA uni 10.4 12.2 R 24 AGC AGT uni 19.5 12.1 S 25 CGG AGG uni 11.4 12 R 26 GGT GGA uni 10.8 16.5 G 27 GGC GGG uni 22.2 16.5 G Note: C-depletion has precedent on U-depletion
Cytidine-depletion combined with Uridine-depletion, with Cytidine taking precedent over Uridine. The reduction of cytidine is combined with exchanging the codon for the highest frequency codon available. This is also applied to codons that would not be changed because of Cytidine or Uridine content. Cytidine or Uridine reduction takes precedent over codon frequency optimization.
TABLE-US-00011 Codon exchange table 2B Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTC TTT uni 20.3 17.6 F 2 TTA TTG uni 7.7 12.9 L 3 CTT CTG uni 13.2 39.6 L 4 CTC CTG uni 19.6 39.6 L 5 CTA CTG uni 7.2 39.6 L 6 ATT ATA uni 16 7.5 I* 7 ATC ATA uni 20.8 7.5 I* 8 GTT GTG uni 11 28.1 V 9 GTC GTG uni 14.5 28.1 V 10 GTA GTG uni 7.1 28.1 V 11 TCT AGT uni 15.2 12.1 S 12 TCC AGT uni 17.7 12.1 S 13 TCA AGT uni 12.2 12.1 S 14 TCG AGT uni 4.4 12.1 S 15 CCT CCA uni 17.5 16.9 P 16 CCC CCA uni 19.8 16.9 P 17 CCG CCA uni 6.9 16.9 P 18 ACT ACA uni 13.1 15.1 T 19 ACC ACA uni 18.9 15.1 T 20 ACG ACA uni 6.1 15.1 T 21 GCT GCA uni 18.4 15.8 A 22 GCC GCA uni 27.7 15.8 A* 23 GCG GCA uni 7.4 15.8 A 24 TAC TAT uni 15.3 12.2 Y 25 CAC CAT uni 15.1 10.9 H 26 CAA CAG uni 12.3 34.2 Q 27 AAC AAT uni 19.1 17 N 28 AAA AAG uni 24.4 31.9 K 29 GAC GAT uni 25.1 21.8 D 30 GAA GAG uni 29 39.6 E 31 TGC TGT uni 12.6 10.6 C 32 CGT AGA uni 4.5 12.2 R 33 CGC AGA uni 10.4 12.2 R 34 CGA AGA uni 6.2 12.2 R 35 CGG AGA uni 11.4 12.2 R 36 AGG AGA uni 12 12.2 R 37 AGC AGT uni 19.5 12.1 S 38 GGT GGA uni 10.8 16.5 G 39 GGC GGA uni 22.2 16.5 G 40 GGG GGA uni 16.5 16.5 G *frequency reduction Note: C-depletion has precedent on U-depletion
Cytidine-depletion combined with Uridine-depletion, with Uridine taking precedent over Cytidine. Also, the exchange aims to respect codon usage frequency, by exchanging high frequency codons with high frequency codons, and exchange low frequency codons with low frequency codons.
TABLE-US-00012 Codon exchange table 2C Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTT TTC uni 17.6 20.3 F 2 TTA CTA uni 7.7 7.2 L 3 CTT CTC uni 13.2 19.6 L 4 ATT ATC uni 16 20.8 I 5 GTT GTA uni 11 7.1 V 6 TCT AGC uni 15.2 19.5 S 7 TCC AGC uni 17.7 19.5 S 8 TCA AGC uni 12.2 19.5 S 9 CCT CCA uni 17.5 16.9 P 10 CCC CCA uni 19.8 16.9 P 11 ACT ACA uni 13.1 15.1 T 12 ACC ACA uni 18.9 15.1 T 13 GCT GCA uni 18.4 15.8 A 14 GCC GCA uni 27.7 15.8 A 15 TAT TAC uni 12.2 15.3 Y 16 CAT CAC uni 10.9 15.1 H 17 AAT AAC uni 17 19.1 N 18 GAT GAC uni 21.8 25.1 D 19 TGT TGC uni 10.6 12.6 C 20 CGT CGA uni 4.5 6.2 R 21 CGC AGA uni 10.4 12.2 R 22 AGT AGC uni 12.1 19.5 S 23 CGG AGG uni 11.4 12 R 24 GGT GGA uni 10.8 16.5 G 25 GGC GGG uni 22.2 16.5 G
Cytidine-depletion combined with Uridine-depletion, with Uridine taking precedent over Cytidine. The reduction of cytidine is combined with exchanging the codon for the highest frequency codon available. This is also applied to codons that would not be changed because of Cytidine or Uridine content. Cytidine and Uridine reduction takes precedent over codon frequency optimization.
TABLE-US-00013 Codon exchange table 2D Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTT TTC uni 17.6 20.3 F 2 TTA CTG uni 7.7 39.6 L 3 TTG CTG uni 12.9 39.6 L 4 CTT CTG uni 13.2 39.6 L 5 CTC CTG uni 19.6 39.6 L 6 CTA CTG uni 7.2 39.6 L 7 ATT ATC uni 16 20.8 I 8 GTT GTG uni 11 28.1 V 9 GTC GTG uni 14.5 28.1 V 10 GTA GTG uni 7.1 28.1 V 11 TCT AGC uni 15.2 19.5 S 12 TCC AGC uni 17.7 19.5 S 13 TCA AGC uni 12.2 19.5 S 14 TCG AGC uni 4.4 19.5 S 15 AGT AGC uni 12.1 19.5 S 16 CCT CCA uni 17.5 16.9 P 17 ccc CCA uni 19.8 16.9 P 18 CCG CCA uni 6.9 16.9 P 19 ACT ACA uni 13.1 15.1 T 20 ACC ACA uni 18.9 15.1 T 21 ACG ACA uni 6.1 15.1 T 22 GCT GCA uni 18.4 15.8 A 23 GCC GCA uni 27.7 15.8 A 24 GCG GCA uni 7.4 15.8 A 25 TAT TAC uni 12.2 15.3 Y 26 CAT CAC uni 10.9 15.1 H 27 CAA CAG uni 12.3 34.2 Q 28 AAT AAC uni 17 19.1 N 29 AAA AAG uni 24.4 31.9 K 30 GAT GAC uni 21.8 25.1 D 31 GAA GAG uni 29 39.6 E 32 TGT TGC uni 10.6 12.6 C 33 CGT AGA uni 4.5 12.2 R 34 CGC AGA uni 10.4 12.2 R 35 CGA AGA uni 6.2 12.2 R 36 CGG AGA uni 11.4 12.2 R 37 AGG AGA uni 12 12.2 R 38 GGT GGA uni 10.8 16.5 G 39 GGC GGA uni 22.2 16.5 G Note: U-depletion takes precedent over C-depletion, which takes precedent over codon optimality.
Guanosine-depletion combined with Uridine-depletion, with Uridine taking precedent over Guanosine. Also, the exchange aims to respect codon usage frequency, by exchanging high frequency codons with high frequency codons, and exchange low frequency codons with low frequency codons.
TABLE-US-00014 Codon exchange table 3 Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTT TTC uni 0.45 0.55 F 2 TTA CTA uni 0.07 0.07 L 3 TTG CTT uni 0.13 0.13 L 4 ATT ATC uni 0.36 0.48 I 5 GTT GTA uni 0.18 0.11 V 6 GTA GTC uni 0.11 0.24 V 7 TCT AGC uni 0.18 0.24 S 8 TCA AGC uni 0.15 0.24 S 9 TCC AGC uni 0.22 0.24 S 10 AGT AGC uni 0.15 0.24 S 11 CCT CCA uni 0.28 0.27 P 12 CCC CCA uni 0.33 0.27 P 13 ACT ACA uni 0.24 0.28 T 14 TAT TAC uni 0.43 0.57 Y 15 CAT CAC uni 0.41 0.59 H 16 AAT AAC uni 0.46 0.54 N 17 AAG AAA uni 0.58 0.42 K 18 GAT GAC uni 0.46 0.54 D 19 CGT CGA uni 0.08 0.11 R 20 CGG CGC uni 0.21 0.19 R 21 CGG CGC uni 0.21 0.19 R 22 AGG AGA uni 0.2 0.2 R 23 AGA CGC uni 0.2 0.19 R
Uridine-depletion without the intention to reduce any other nucleotide, although this might happen to a minor extent. Also, the exchange aims to respect codon usage frequency, by exchanging high frequency codons with high frequency codons, and exchange low frequency codons with low frequency codons.
TABLE-US-00015 Codon exchange table 4A Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTT TTC uni 17.6 20.3 F 2 TTG CTC uni 12.9 19.6 L 3 CTT CTC uni 13.2 19.6 L 4 ATT ATC uni 16 20.8 I 5 GTT GTC uni 11 14.5 V 6 GCT GCA uni 18.4 15.8 A 7 ACT ACA uni 13.1 15.1 T 8 CCT CCC uni 17.5 19.8 P 9 TCT TCC uni 15.2 17.7 S 10 TAT TAC uni 12.2 15.3 Y 11 CAT CAC uni 10.9 15.1 H 12 AAT AAC uni 17 19.1 N 13 GAT GAC uni 21.8 25.1 D 14 TGT TGC uni 10.6 12.6 C 15 CGT CGA uni 4.5 6.2 R 16 AGT AGC uni 12.1 19.5 S 17 GGT GGA uni 10.8 16.5 G
Uridine-depletion without the intention to reduce any other nucleotide, although this might happen to a minor extent. The reduction of uridine is combined with exchanging the codon for the highest frequency codon available. This is also applied to codons that would not be changed because of Uridine content. Uridine reduction takes precedent over codon frequency optimization.
TABLE-US-00016 Codon exchange table 4B Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 1 TTT TTC uni 17.6 20.3 F 2 TTA CTG uni 7.7 39.6 L** 3 TTG CTG uni 12.9 39.6 L 4 CTT CTG uni 13.2 39.6 L 5 CTC CTG uni 19.6 39.6 L** 6 CTA CTG uni 7.2 39.6 L 7 ATT ATC uni 16 20.8 I 8 ATA ATC uni 7.5 20.8 I 9 GTT GTG uni 11 28.1 V 10 GTC GTG uni 14.5 28.1 V** 11 GTA GTG uni 7.1 28.1 V 12 TCT AGC uni 15.2 19.5 S 13 TCC AGC uni 17.7 19.5 S** 14 TCA AGC uni 12.2 19.5 S 15 TCG AGC uni 4.4 19.5 S 16 AGT AGC uni 12.1 19.5 S 17 CCT CCC uni 17.5 19.8 P 18 CCG CCC uni 6.9 19.8 P 19 ACT ACC uni 13.1 18.9 T 20 ACG ACC uni 6.1 18.9 T 21 GCT GCC uni 18.4 27.7 A 22 GCA GCC uni 15.8 27.7 A 23 GCG GCC uni 7.4 27.7 A 24 TAT TAC uni 12.2 15.3 Y 25 CAT CAC uni 10.9 15.1 H 26 CAA CAG uni 12.3 34.2 Q 27 AAT AAC uni 17 19.1 N 28 AAA AAG uni 24.4 31.9 K 29 GAT GAC uni 21.8 25.1 D 30 GAA GAG uni 29 39.6 E 31 TGT TGC uni 10.6 12.6 C 32 CGT AGA uni 4.5 12.2 R 33 CGC AGA uni 10.4 12.2 R 34 CGA AGA uni 6.2 12.2 R 35 CGG AGA uni 11.4 12.2 R 36 AGG AGA uni 12 12.2 R 37 GGT GGC uni 10.8 22.2 G 38 GGG GGC uni 16.5 22.2 G 38 GGA GGC uni 16.5 22.2 G **C-depletion
Cytidine-depletion without the intention to reduce Uridine, although this might happen to a minor extent. Also, the exchange aims to respect codon usage frequency, by exchanging high frequency codons with high frequency codons, and exchange low frequency codons with low frequency codons. This codon exchange table is used to obtain a dose-effect of C-depletion for comparison to C2-depletion (codon exchange table 1A)
TABLE-US-00017 Codon exchange table 5 Freq human Freq human Original Swap codon usage codon usage Amino codon codon Direction (original) (swap) acid 9 CCC CCA uni 19.8 16.9 P 10 ACC ACA uni 18.9 15.1 T 11 GCC GCA uni 27.7 15.8 A* 12 CGC CGG uni 10.4 11.4 R 13 GGC GGA uni 22.2 16.5 G *Reduced frequency Note: Rules are to change every C to A or Gor U. Reducing C takes precedent on codon frequency.