DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 12-19 are withdrawn from consideration.
Claims 1-11 are examined on the merits.
Election/Restrictions
Claims 12-19 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 09/12/2025.
Applicant’s election without traverse of Group I corresponding to claims 1-11, in the reply filed on 09/12/2025 is acknowledged.
Priority
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in parent Application No.PCT/JP2019/051241, filed on 12/26/2019.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-3, 5, 7, 11 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kanda et al. (“Kanda”, Biochemical and Biophysical Research Communications, cited as reference NPL9 in IDS filed 12/16/2022).
Regarding claims 1, 5, 7, Kanda teaches methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids. With 10 mM Mg2+, the translation efficiency was higher than that with the other tested concentration, and the misreading efficiency was low. The protease mRNA was translated in the presence of an antisense DNA-treated tRNA mixture and 2-naphthylalanyl- and/or p-phenylazophenylalanyl-tRNA. Kanda teaches a method for substitution of multiple positions with pre-charged amino acids via cell-free protein synthesis. Previous studies show that antisense DNAs are useful for inactivating tRNAAsp and tRNAPhe in E. coli crude tRNA mixture. tRNAs in the S30 extract can be removed without essential damage in the ribosomal activity by a treatment with an immobilized RNase A. Unnatural amino acids (Fig. 1 (A) and (B)) pre-charged on artificial tRNAs (Fig. 1 (C)) can be introduced at multiple sites of a variant of the HIV-1 protease protein (Fig. 2), substituting the original Asp and/or Phe residues (it reads on first and second tRNA and artificial tRNA) (e.g., paragraph 2nd, right column, page 1136; Fig. 1A-C [see below]; Fig. 2).
Fig. 1 (Structures of the unnatural amino acids and tRNAs used in the present study. (A) 2-Naphthylalanine (2-napAla); (B)
p-phenylazophenylalanine (azoAla); (C) tRNA-GUC and tRNA-GAA
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Fig. 2 (Amino acid sequence encoded by the HIV-1 protease mRNA. Positions of Asp and Phe residues are indicated
by the underlined bold letters. The T7 z tag peptide is indicated).
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Kanda teaches introduction of unnatural amino acids into HIV-1 protease. The protease mRNA was translated in the presence of 10 mM Mg21, tRNAMixΔΔDF, and two unnatural aminoacyl-tRNAs. Full-length product was efficiently synthesized when one of the 2-napAlatRNAs
and one of the azoAla-tRNAs were used (it reads on two unnatural amino acids) (lanes 3–6) (e.g., paragraph 2nd, right column, page 1138; Fig. 6).
Fig. 6
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Kanda teaches 32C, 33U, 37G and 38A positions in tRNA (it reads on option (6)) (e.g., Fig. 1C).
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Kanda teaches methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids (e.g., abstract; Fig. 1, Fig. 6). Asp (codons: 5’ GAU, GAC 3’ and their anticodons 5’ GUC, AUC 3’). Phe (codons: 5’ UUU, UUC 3’ and their anticodons: 5’ GAA, AAA 3’), as indicated in Fig. 1C. Kanda teaches production of the mistranslated full-length protein may depend on tRNA species that is inactivated. It is expected that proteins with unnatural amino acid residues could be more flexibly prepared by the sense codon-dependent method (e.g. paragraph 1st, column left, page 4). Kanda teaches that the HIV-1 protease contains leucine and glutamine residues (e.g., Fig. 2). Leu (codons: 5’ UUA, UUG 3’ and their anticodons 5’ CAA, UAA 3’). Glu (codons: 5’ GAA, GAG 3’ and their anticodons: 5’ CUC, UUC 3’). Therefore, it can be inferred by a person skilled in the art, that the E. coli-derived tRNA described by Kanda includes a tRNA to which leucine is bound and has UAA or CAA as an anticodon ("Leu-tRNA-UAA" or "Leu-tRNA-CAA," respectively), and a tRNA to which glutamate is bound and has UUC or CUC as an anticodon ("Glu-tRNA-UUC" or "Glu-tRNA-CUC," respectively). Therefore, the first tRNA and the second tRNA in Claim 1 of the instant application correspond to 2-napAla-tRNA-GUC and Glu-tRNA-UUC, 2-napAla-tRNA-GAA and Leu-tRNA-CAA, azoAla-tRNA-GUC and Glu-tRNA-CUC, or azoAla-tRNA-GAA and Leu-tRNA-UAA.
Regarding claim 2, Kanda teaches that the HIV-1 protease contains leucine and glutamine residues (e.g., Fig. 2). Leu (codons: 5’ CUU 3’ and their anticodons 5’ CAA 3’). Glu (codons: 5’ GAG 3’ and their anticodon: 5’ CUC 3’) (it reads Leu first tRNA, first base anticodon “A”; Glu second tRNA, first letter anticodon “C”).
Regarding claim 3, Kanda teaches production of the mistranslated full-length protein may depend on tRNA species that is inactivated. It is expected that proteins with unnatural amino acid residues could be more flexibly prepared by the sense codon-dependent method (e.g. paragraph 1st, left column, page 1139). Kanda teaches that the HIV-1 protease contains 20 different amino acid residues (MASTGQEFPILWRVKDYCHN) (e.g., Fig. 2). Therefore, it can be inferred by a person skilled in the art, that the E. coli-derived tRNA described by Kanda may include a tRNA bound to an unnatural amino acid with different anticodons. As such, leucine (L) bound to tRNA has UAA or CAA as an anticodon ("Leu-tRNA-UAA" or "Leu-tRNA-CAA," respectively); glutamate (E) bound to tRNA has UUC or CUC as an anticodon ("Glut-RNA -UUC" or "Glu-tRNA-CUC," respectively); proline (P) attached to a tRNA has CGG, UGG, GGG, AGG as an anticodon ("Pro-tRNA-CGG", "Pro-tRNA-UGG", "Pro-tRNA-GGG", "Pro-tRNA-AGG", respectively); glycine (G) attached to a tRNA has CCC, UCC, GCC, ACC as an anticodon ("Gly-tRNA-CCC", "Gly-tRNA-UCC", "Gly-tRNA-GCC", "Gly-tRNA-ACC", respectively); cysteine (C) attached to tRNA has GCA, ACA as an anticodon ("Cys-tRNA-GCA" or "Cys-tRNA-ACA," respectively). A similar analysis can be done for all the amino acids. Therefore, the first tRNA and the second tRNA in Claim 3 of the instant application correspond to 2-napAla-tRNA-GUC and Glu-tRNA-UUC, 2-napAla-tRNA-GAA and Leu-tRNA-CAA, azoAla-tRNA-GUC and Glu-tRNA-CUC, or azoAla-tRNA-GAA and Leu-tRNA-UAA.
Regarding claim 11, Kanda teaches production of the mistranslated full-length protein may depend on tRNA species that is inactivated. It is expected that proteins with unnatural amino acid residues could be more flexibly prepared by the sense codon-dependent method (e.g. paragraph 1st, left column, page 1139). Kanda teaches that the HIV-1 protease contains 20 different amino acid residues (MASTGQEFPILWRVKDYCHN) (e.g., Fig. 2). Kanda teaches two unnatural aminoacyl-tRNAs, 2-napAlatRNAs (2-napAla-tRNA-GUC, 2-napAla-tRNA-GAA) and azoAla-tRNAs (azoAla-tRNA-GUC and azoAla-tRNA-GAA) with anticodons corresponding to either aspartic acid (GUC) or phenylalanine (GAA) (e.g., paragraph 2nd, right column, page 1138). Therefore, one skilled in the art could use an artificial tRNA bound to an unnatural amino acid to replace aspartic acid, phenylalanine or any amino acid following the method of Kanda.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim 4, 6, 9 are rejected under 35 U.S.C. 103 as being unpatentable over Kanda et al. (“Kanda”, Biochemical and Biophysical Research Communications, cited as reference NPL9 in IDS filed 12/16/2022) in view of Murakawi et al. (“Murakawi”, US 2018/0298390 A1, cited as reference US2 on IDS filed 12/16/2022).
Kanda does not teach a chimeric tRNA body as required by the instant claims 4, 6, 9. However, this is cured by Murakawi.
Regarding claims 4, 6, 9, Murakawi teaches translation system that can express polypeptide according to a genetic code different from the universal genetic code (e.g., paragraph 0011). Murakawi teaches interchanged codons for these amino acids in a gene encoding a protein and interchanged the relations between the anticodons and the amino acids in the aminoacyl-tRNAs correspondingly and succeeded in expressing polypeptides having predetermined amino acid sequences according to a new genetic code different from the universal genetic code (e.g., paragraph 0013). Murakawi teaches the universal genetic code and codon-shuffled genetic codes. (A) illustrates the universal genetic code. tRNAAla, tRNALeu, and tRNASer have the original anticodon loops. (B) illustrates codon-shuffled genetic code 1. The CUU and CUC codons are assigned to Ser and the UCU and UCC codons are assigned to Leu and also, chimeric tRNALeu and chimeric tRNASer in which the anticodon loops are interchanged are used. (C) illustrates codon-shuffled genetic code 2. The GCU and GCC codons are assigned to Ser, the CUU and CUC codons are assigned to Ala, and the UCU and UCC codons are assigned to Leu and also chimeric tRNAAla, chimeric tRNALeu, and chimeric tRNASer in which the anticodon loops are interchanged are used (e.g., paragraph 0082; Fig. 2 [see below]).
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Murakawi teaches the translation system comprising: a template nucleic acid having a sequence modified from a nucleic acid sequence encoding the polypeptide by interchanging codons encoding at least 2 amino acids selected from serine, leucine, and alanine; and at least either (i) or (ii): (i) an aminoacyl-tRNA having an anticodon to codon after interchange and to which an amino acid encoded by a codon before the interchange is bound; (ii) a tRNA having an anticodon to a codon after interchange and recognized by an aminoacyl-tRNA synthetase corresponding to an amino acid encoded by a codon before the interchange or a nucleic acid encoding the tRNA, the amino acid, and the aminoacyl-tRNA synthetase or a nucleic acid encoding the aminoacyl-tRNA synthetase (e.g., paragraphs 0074-0078).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to use the chimeric tRNALeu and chimeric tRNASer in which the anticodon loops are interchanged taught by Murakawi, an incorporate in the cell-free protein synthesis method for substitution of multiple positions with pre-charged amino acids via cell-free protein synthesis taught by Kanda.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to do so in order to develop a method of cell-free protein synthesis, driven by a crude extract from an organism, for the preparation of proteins containing unnatural amino acids at specific positions using chimeric tRNAs.
Claim 8, 10 is rejected under 35 U.S.C. 103 as being unpatentable over Kanda et al. (“Kanda”, Biochemical and Biophysical Research Communications, cited as reference NPL9 in IDS filed 12/16/2022) and Murakawi et al. (“Murakawi”, US 2018/0298390 A1, cited as reference US2 on IDS filed 12/16/2022) as applied to claims 1-7, 9, 11 above, and further in view of Kariyuki et al. (“Kariyuki”, US 20150080549 A1, cited as reference US2 on IDS filed 09/12/2025) and Sprinzl et al. (“Sprinzl”, Nucleic Acid Research, 1987).
Kanda and Murakawi does not teach SEQ ID NO 255 as required by instant claim 8. Kanda and Murakawi does not teach a chimeric tRNA body that has 32U, 33U, 37G, and 38U as required by instant claims 8 and 10. However, this is cured by Kariyuki and Sprinzl.
Kariyuki teaches methods for synthesizing cyclized peptides libraries by the combination of a novel method for translation and posttranslational chemical modification. Furthermore, methods for synthesizing libraries of peptide compounds further having a linear portion that increases the potential for obtaining druglike peptides having the activity of interest (e.g., paragraph 0020). Kariyuki teaches that unnatural amino acid that can be used in translational synthesis (e.g., paragraph 0461). Kariyuki teaches SEQ ID NO 124 that has 100% homology with SEQ ID NO 255 of the instant claim (e.g., page 446).
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Sprinzl teaches the numbering system derived from yeast tRNAPhe tRNA structure (where positions 34, 35 and 36 correspond to the anticodon) (e.g., paragraph 1st, page r53; Fig. 1).
Fig. 1 Numbering of Nucleotides in tRNAs
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Sprinzl teaches a compilation of 413 tRNA sequences and 665 sequences of tRNA genes [see Tables below] so far published covering the literature to the end of 1986 (e.g., paragraph 1st, page r53). Sprinzl teaches that the title of each sequence contains a one letter code for the particular amino acid depicting the specificity of tRNA, a three digit number, the anticodon sequence i-n unmodified form and name of the organism from which the tRNA originates (e.g., paragraph 2nd, page r53). Sprinzl teaches tRNA genes comprising 32T, 33T, 37G, 38T (it reads on 32U, 33U, G37, 38U). See tables blow:
[AltContent: rect]Sprinzl teaches tRNA for Alanine A493, human mitochondria (with anticodon TGC) (e.g., r118).
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Sprinzl teaches tRNA gene for Glutamine, Q335 (Nicotiana tabacum), Q410 (aspergillus) (with anticodon TTG) (e.g., r130)
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Sprinzl teaches tRNA gene for Histidine, H417 (saccharomyces cerevisiae), H425 (torulopsis glab.) H468 (with anticodon GTG) (drosophila yakuba) (e.g., r138)
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Sprinzl teaches tRNA gene for Leucine, L250 (E. coli), L251 (E. coli) (with anticodon CAG) (e.g., r142)
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Sprinzl teaches tRNA gene for Proline, P335 (nicotiana tabacum), P410 (with anticodon TGG) (aspergillus) (e.g., r158)
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It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to use the tRNA structure with SEQ ID NO 124 taught by Kariyuki, and tRNA numbering system derived from yeast tRNAPhe comprising 32U, 33U, 37G, 38U with anticodon different than GCG or CCG as taught by Sprinzl an incorporate in the method for substitution of multiple positions with pre-charged amino acids via cell-free protein synthesis using chimeric tRNA taught by Kanda and Murakawi .
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to do so in order to develop a method of cell-free protein synthesis, driven by a crude extract from and organism, for the preparation of proteins containing unnatural amino acids at specific positions using chimeric tRNAs comprising 32U, 33U, 37G, 38U in the tRNA structure.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JULIO GOMEZ RODRIGUEZ whose telephone number is (571)270-0991. The examiner can normally be reached Monday - Friday 8:00 am - 5:00 pm.
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/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/J. E. ANGELL, Ph.D./Primary Examiner, Art Unit 1637