The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This office action is in response to Applicants’ amendments/remarks received May 19, 2026.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claims 2, 21-28 are canceled. Claims 17-20 are withdrawn. Claims 1, 3-16, 29, to SEQ ID NO: 2, are under consideration.
Priority: This application is a 371 of PCT/US2021/026202, filed April 7, 2021, which claims benefit of provisional applications 63/008098, filed April 10, 2020, and 63/147352, filed February 9, 2021.
Objections and Rejections
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.
Claims 1, 3, 8-16 are rejected under 35 U.S.C. 103 as being unpatentable over Dominguez et al. (1998 Internatl Microbiol 1:131-142; previously cited), evidenced by Prado-Gonzalez et al. (Sequence submission of Prado-Gonzalez et al. Thesis 1993; previously cited), and in view of Darvishi et al. (2018 Applied Microbiology and Biotechnology 102:5925-5938; previously cited).
Dominguez et al. disclose non-conventional yeasts, including Yarrowia lipolytica (Y. lipolytica), for heterologous protein production (at least p. 131). Dominguez et al. disclose homologous promoters in the yeast Y. lipolytica for the expression of heterologous protein include XPR2 and MTP I/II (p. 133 Table 2). Dominguez et al. disclose developing vectors for Y. Lipolytica, the vector comprising a promoter and a polylinker sequence (p. 136-138, Fig. 2). Therefore, it would have been obvious to one of ordinary skill that a vector can be developed to comprise a MTP I/II promoter and a polylinker sequence.
While Dominguez et al. do not explicitly teach the nucleic acid sequence for the promoter to MTP I/II, it is noted that the MTP I/II promoter comprises a nucleic acid sequence having at least 95% sequence identity with the recited promoter sequence comprising instant SEQ ID NO: 2, as submitted in the thesis of Prado-Gonzalez et al. See below:
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987
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810
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572
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Therefore, Dominguez et al. can be deemed to disclose developing a vector (or transcription element) comprising a Y. lipolytica promoter and a polylinker, where known Y. lipolytica promoters include MTP I/II promoter, which appears to be the same promoter of instant SEQ ID NO: 2. Dominguez et al. et al. do not teach upstream activation sequences (UASs) operably linked to the promoter.
Darvishi et al. disclose Y. lipolytica is an important industrial host for the production of recombinant proteins and chemicals (p. 5925). Darvishi et al. disclose that promoters are the key elements in controlling gene expression and that the strength of a promoter is determined by factors including core promoter, TATA box, proximal promoter sequence, and upstream activating sequences (UAS) (p. 5926). Darvishi et al. disclose strong hybrid promoters are engineered by fusing UAS to the promoter; the fusion of between 1 and 32 tandem UAS sequences from pLEUm and 8-16 copies of UAS from TEF1 increased the mRNA and protein expression levels (p. 5926). Darvishi et al. disclose for example, mRNA level was 400-fold higher for hybrid promoter UAS1B24-LEUm in comparison with the core promoter pLEUm (p. 5926).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further operably link at least 16 UASs to the promoter MTP I/II in the vector developed to comprise promoter MTP I/II and a polylinker of Dominquez et al. noted above, to thereby arrive at the claimed transcription element (instant claims 1, 3, 8, 9-16). The motivation to do is given by the prior art Darvishi et al., which disclose strong hybrid promoters are engineered by fusing a core promoter with a plurality of UASs (p. 5926). One of ordinary skill would have a reasonable expectation of success because elements for developing vectors and transcription elements for heterologous protein expression are known and available in the prior art, and the prior art has identified known Y. lipolytica promoters, including the instant promoter sequence.
Further, regarding instant claim 3, Darvishi et al. disclose fusing a plurality of UASs, i.e. UAS1B24, to a promoter increased mRNA level by 400-fold (p. 5926). Therefore, Darvishi et al. can be deemed to disclose that each of the UAS is identical.
Regarding instant claim 8, Darvishi et al. disclose that many of the genes encoding central metabolic enzymes in Y. lipolytica contain introns at the beginning of the gene, where including these introns improve expression of heterologous genes (p. 5926). Darvishi et al. disclose that an intron-containing translation elongation factor-1α (TEF1intron) promoter exhibited a 17- and 5-fold increase in gene expression over intron-less TEF1 and HP4D promoters (p. 5926). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further include a TEF1intron in the vector developed to comprise promoter MTP I/II, a polylinker, and at least 16 UASs of Dominquez et al. in view of Darvishi et al. noted above, where the TEF1intron is positioned between the promoter and polylinker as an obvious design choice. The motivation to do so is given by the prior art Darvishi et al., which disclose including Y. lipolytica introns, such as TEF1 intron, improve expression of heterologous genes. One of ordinary skill would have a reasonable expectation of success because elements for developing vectors and transcription elements for heterologous protein expression are known and available in the prior art.
Regarding instant claim 10, as noted above, Dominguez et al. disclose the vector comprises the Y. lipolytica promoter operably linked to a polylinker sequence. MPEP 2144.04 notes that the duplication of part is obvious. Therefore, it would be obvious that the vector developed to comprise the Y. lipolytica promoter MTP I/II can be flanked on each end by a polylinker sequence as an obvious design choice.
Regarding instant claims 11, 13, Dominguez et al. disclose integrating a LEU2 region (p. 136-137).
Regarding instant claims 12, 14, Dominguez et al. disclose that the usual transforming vectors for yeast are hybrids between yeast-derived and bacterial sequences, because plasmids can be amplified and isolated with greater ease from E. coli, harbor an ori, and also have a sequence conferring resistance against a specific antibiotic (p. 133). Therefore, it would be obvious to one of ordinary skill that the vector comprising the Y. lipolytica promoter MTP I/II can further comprise an antibiotic resistance marker (instant claim 12) and/or an E. coli replication region (instant claim 14).
Regarding instant claims 15-16, as noted above, Dominguez et al. disclose Y. lipolytica is a system for heterologous protein production (p. 131). Therefore, it would be obvious to one of ordinary skill that the vector comprising the Y. lipolytica promoter MTP I/II of Dominguez et al. can be operably linked to a heterologous coding sequence (instant claim 15) and it would be further obvious that a Y. lipolytica host cell can comprise the vector (instant claim 16).
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive. The reasons for maintaining the 103 rejection are noted above and noted herein.
Applicants have amended instant claim 1 to recite “at least 16 upstream activation sequences (UASs) operably linked to said promoter sequence.” Applicants assert that the examiner has not established a prima facie case of obviousness. Applicants assert that even if the examiner has established a prima facie case of obviousness, Applicants have rebutted the examiner’s prima facie case of obviousness by demonstrating that the amended claims have unexpected results compared to the combined teachings of Dominguez et al./Darvishi et al. Applicants assert that example 4 of the instant application compares the effects of UAS copy number on the strength of promoters with and without addition of copper. Applicants assert that as shown in Fig. 5, including at least 16 UASs demonstrated surprisingly non-linear improvement compared to the inclusion of fewer UASs. Applicants assert that these effects are unexpected and not predictable from the combined teachings of Dominguez et al./Darvishi et al.
Applicants’ remarks are not persuasive. In this instance, Darvishi et al. disclose that strong hybrid promoters are engineered by fusing UAS to the promoter; the fusion of between 1 and 32 tandem UAS sequences from pLEUm and 8-16 copies of UAS from TEF1 increased the mRNA and protein expression levels (p. 5926). Darvishi et al. disclose for example, mRNA level was 400-fold higher for hybrid promoter UAS1B24-LEUm in comparison with the core promoter pLEUm (p. 5926). Therefore, Applicants’ remarks that including at least 16 UASs demonstrated surprising improvement compared to the inclusion of fewer UASs are not persuasive because the prior art has already disclosed and recognized that including a plurality of UASs enhances promoter strength, including the recited at least 16 UASs. Darvishi et al. has disclosed that promoter strength can be fine-tuned by engineering of the TATA box sequence, core promoter, and upstream activating sequences, citing Hussain et al. 2015 (p. 5926). Hussain et al. disclose that increasing the number of (UAS) repeats monotonically and cooperatively increases the transcription from the downstream core promoter (p. 215) and that this modular architecture suggests that promoter strength and induction properties should be predictably engineerable using defined UASs (p. 215).
Therefore, Applicants’ remarks that the effects of at least 16 UASs on the promoter are unexpected and not predictable are not found persuasive. In this instance, it would have been expected and predictable that the inclusion of at least 16 UASs enhances promoter strength and increases protein expression.
Claims 1, 3, 8-16, 29 are rejected under 35 U.S.C. 103 as being unpatentable over Dominguez et al. (1998 Internatl Microbiol 1:131-142; previously cited) in view of Darvishi et al. (2018 Applied Microbiology and Biotechnology 102:5925-5938; previously cited) and Madzak et al. (US 6083717). The teachings of Dominguez et al. in view of Darvishi et al. over at least instant claims 1-3, 8-16 are noted above.
Regarding instant claim 29, Darvishi et al. disclose fusing a plurality of UASs, i.e. UAS1B24, to a promoter increased mRNA level by 400-fold (p. 5926). Madzak et al. disclose known UAS, including UAS1B, comprising SEQ ID NO: 4, which has 100% sequence identity to instant SEQ ID NO: 12 (at least col. 3 lines 53-67). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate at least 16 UASs, each comprising SEQ ID NO: 4 of Madzak et al., to the promoter MTP I/II in the vector developed to comprise promoter MTP I/II, a polylinker, and at least 16 UASs of Dominquez et al. in view of Darvishi et al. noted above, to thereby arrive at the claimed transcription element. One of ordinary skill would have a reasonable expectation of success because elements for developing vectors and transcription elements for heterologous protein expression are known and available in the prior art.
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive. New claim 29 is rejected under 35 U.S.C. 103 for the reasons noted above. The reasons for maintaining Dominguez et al./Darvishi et al. are the same as noted above.
Claims 1, 3, 4-6, 8-16 are rejected under 35 U.S.C. 103 as being unpatentable over Dominguez et al. (1998 Internatl Microbiol 1:131-142; previously cited) in view of Darvishi et al. (2018 Applied Microbiology and Biotechnology 102:5925-5938; previously cited), Szymczak et al. (2005 Expert Opin Biol Ther 5(5):627-638; previously cited) and O’Shea et al. (US 20180355379; previously cited). The teachings of Dominguez et al. in view of Darvishi et al. over at least instant claims 1-3, 8-16 are noted above.
Regarding instant claim 4, Szymczak et al. disclose utilizing 2A peptides to design vectors to express multiple genes (p. 627). Szymczak et al. disclose that the advantages of using 2A peptide sequences include their small size and ability for efficient coexpression of genes that are placed between them; genes placed downstream of different 2A peptide sequences are able to induce higher levels of expression when compared with placement following IRES (p. 631). Due to their small size and divergent N-terminal sequence, multiple 2A peptide sequences can be used in vectors (p. 632). Szymczak et al. disclose functional 2A-peptide sequences used in vector construction (p. 631 Table 2). O’Shea et al. also disclose 2A peptides allows for expression of multiple proteins (paragraphs 0146-0147). O’Shea et al. disclose exemplary 2A peptides, including the 2A peptides of Szymczak et al. (paragraphs 0147-0148), where the 2A peptides are modified to include Gly-Ser-Gly at the N terminus to improve cleavage efficiency, and where modified P2A sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 18) and modified T2A sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 21) have 100% sequence identity with instant SEQ ID NO: 13 and 14, respectively. Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further include a 2A peptide coding nucleic acid sequence downstream of the polylinker in the vector developed to comprise promoter MTP I/II, a polylinker, and at least 16 UASs of Dominquez et al. in view of Darvishi et al. noted above. The motivation to do so is given by the prior art Szymczak et al. and/or O’Shea et al., which disclose the advantages of utilizing 2A peptides in vector design to express multiple proteins. One of ordinary skill would have a reasonable expectation of success because elements for developing vectors and transcription elements for heterologous protein expression are known and available in the prior art.
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive. The reasons for maintaining Dominguez et al./Darvishi et al. are the same as noted above.
Claims 1, 3, 4-6, 7, 8-16 are rejected under 35 U.S.C. 103 as being unpatentable over Dominguez et al. (1998 Internatl Microbiol 1:131-142; previously cited) in view of Darvishi et al. (2018 Applied Microbiology and Biotechnology 102:5925-5938; previously cited), Szymczak et al. (2005 Expert Opin Biol Ther 5(5):627-638; previously cited), O’Shea et al. (US 20180355379; previously cited), and Cesaratto et al. (2016 Journal of Biotechnology 231:239-249; previously cited). The teachings of Dominguez et al., Darvishi et al., Szymczak et al., and O’Shea et al. over at least instant claims 1, 3, 4-6, 8-16 are noted above.
Regarding instant claim 7, Cesaratto et al. disclose Tobacco Etch Virus protease (TEVp) is an efficient and specific protease that has become a valuable biotechnological tool (p. 239). Cesaratto et al. disclose TEVp has often been used to facilitate stoichiometric expression of multiple genes in bacteria, yeast, plant, and mammalian cells (p. 244). Cesaratto et al. disclose that peptide 2A, depending on the upstream protein, may not result in 1:1 stoichiometric expression of protein subunits (p. 244). Cesaratto et al. disclose that TEVp stoichiometry, instead is 1:1 (p. 244). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further incorporate a nucleic acid sequence encoding a TEVp in vector developed to comprise promoter MTP I/II, a polylinker, at least 16 UASs, and a plurality of 2A peptide coding nucleic acid sequences of Dominguez et al., Darvishi et al., Szymczak et al., and O’Shea et al. noted above. The motivation to do so is given by the prior art Cesaratto et al., which disclose TEVp is an efficient tool for facilitating stoichiometric expression of multiple genes in various host cells, including yeast. One of ordinary skill would have a reasonable expectation of success because elements for developing vectors and transcription elements for heterologous protein expression are known and available in the prior art.
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive. The reasons for maintaining Dominguez et al./Darvishi et al. are the same as noted above.
No claim is allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Marsha Tsay whose telephone number is (571)272-2938. The examiner can normally be reached M-F.
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/Marsha Tsay/Primary Examiner, Art Unit 1656
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