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 .
DETAILED ACTION
Claims 1-4, 10-12, 18, 22, 24, 26, 33-36, 38, 44-47 are pending.
This application, filed on 12/22/2023, claims benefit of 63/478,493 filed on 1/4/2023 and 63/477,133 filed on 12/23/2022. The effective filing date of the current application is December 23, 2022.
Information Disclosure Statement
The information disclosure statements filed on 3/11/2024 complies with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. All references were considered.
The information disclosure statement filed 4/27/2026 fails to comply with the provisions of 37 CFR 1.98(a)(4) because it lacks the appropriate size fee assertion. It has been placed in the application file, but the information referred to therein has not been considered as to the merits.
Election/Restrictions
Applicant’s election of Group I, claims 1-4, 10-12, 18, 22, 24, 26 and 33, in the reply filed on April 27, 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 34-36, 38 and 44-47 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on April 27, 2026. Claims 1-4, 10-12, 18, 22, 24, 26 and 33 are under examination herein.
Claim Rejections - 35 USC § 102
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 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624). The rejection of claim 1 is further evidenced by Puchart et al. (“Xylanases of glycoside hydrolase family 30 – An overview”, Biotechnology Advances, 2021, Vol. 47, article 107704, 16 pages).
Regarding claim 1, von der Heyde teaches a P. torridus intein inserted before amino acid residue Thr75 of a hemicellulase reporter enzyme from Pseudoalteromonas arctica (abstract). von der Heyde teaches intein-xylanase constructs (p.7621, Fig. 5A). As evidenced by Puchart, xylanases are glycoside hydrolases, and are part of the glycoside hydrolase families 10 and 11, and new family GH30 (abstract).
Regarding claim 2, von der Heyde teaches that variants including the insertion of a functional intein into the Thr75 insertion site in Xyn8 did not show xylanase activity on plate assays (p.7620, 2nd column 2nd paragraph).
Regarding claim 3, von der Heyde teaches that xylanase derived from T75 intein excision recovered activity when tested on plate assays (Fig. 5d; p.7620 1st column last paragraph).
Claim Rejections - 35 USC § 103
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624) as applied to claim 1 above, and further in view of Srivastava, Smriti ed. (“Industrial Applications of Glycoside Hydrolases”, 2020, Springer; eBook Srivastava editor).
The teachings of von der Heyde et al. are discussed above.
Regarding claim 4, von der Heyde does not teach wherein the glycosyl hydrolase is an O-GlcNAcase (OGA).
However, Shrivastava identifies glycoside hydrolases (i.e. glycosyl hydrolase) as including cellulase, amylases, xylanases, arabinases and others (p.3, 1st paragraph). Shrivastava further teaches enzyme O-GlcNAcase which is involved in removal of N-acetylglucosamine groups from cytoplasmic and nuclear located serine and threonine residues (p.4 – Occurrence and Importance).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the xylanase taught by von der Heyde with O-GlcNAcase taught by Shrivastava, because Shrivasta teaches that both xylanases and O-GlcNAcase are glycoside hydrolases. One of ordinary skill in the art would reasonably expect that replacing one known glycoside hydrolase (xylanase) with another (O-GlcNAcase) would predictably result in a glycosyl hydrolase enzyme comprising an intein, because it would amount to a simple substitution of one known enzyme for another, and it was known in the art at the time of invention that enzymes could be engineered to comprise inteins.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624) as applied to claim 1 above, and further in view of GenBank (accession number XP_033040761, published in GenBank on March 28, 2020).
The teachings of von der Heyde are discussed above.
Regarding claim 10, von der Heyde does not teach wherein the glycosyl hydrolase comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:107.
However, GenBank teaches a protein O-GlcNAcase from Trachypithecus francoisi (title). The sequence of GenBank is 99% identical to instant SEQ ID NO:107.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the xylanase taught by von der Heyde with the O-GlcNAcase taught by GenBank. Each of von der Heyde and GenBank teach glycosyl hydrolase enzymes. One of ordinary skill in the art would reasonably expect that replacing one known glycosyl hydrolase enzyme with another would predictably result in a glycosyl hydrolase comprising the desired amino acid sequence homology, because an O-GlcNAcase glycosyl hydrolase enzyme comprising an amino acid sequence at least 99% identical to instant SEQ ID NO:107 was known in the art at the time of invention.
Claims 11-12 are rejected under 35 U.S.C. 103 as being unpatentable over von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624) as applied to claim 1 above, and further in view of Peck et al. (“Directed Evolution of a Small-Molecule-Triggered Intein with Improved Splicing Properties in Mammalian Cells”, Chemistry & Biology, 2011, Vol. 18, Issue 5, pp. P619-630).
The teachings of von der Heyde are discussed above.
Regarding claims 11 and 12, von der Heyde does not teach wherein the intein is a ligand-dependent intein (claim 11) or wherein the ligand-dependent intein is excised from the glycosyl hydrolase upon binding of the ligand (claim 12).
However, Peck teaches inteins that splice efficiently in Saccharomyces cerevisiae in the presence of the cell-permeable small molecule 4-hydroxytamoxifen (4-HT) (abstract). Peck teaches genetically inserting the 4-HT-dependent intein in place of Cys108 of green fluorescent protein (GFP), which abolishes fluorescence until splicing takes place (p.621, 1st column – Evolution Scheme for Improved 4-HT-Dependent Inteins). Peck teaches that during positive screens for intein splicing activity, cells that exhibited GFP fluorescence in the presence of 4-HT were collected, and during negative screens cells that remained nonfluorescent in the absence of 4-HT were collected (p.621, 1st column – Evolution Scheme for Improved 4-HT-Dependent Inteins). Peck teaches that small molecule-triggered inteins offer single protein specificity and other desirable features as an approach to control protein function in cells post-translationally (p.627, 2nd column – Significance).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the intein taught by von der Heyde with the 4-HT-dependent intein taught by Peck in the xylanase of von der Heyde, because Peck teaches that small molecule-triggered inteins offer single protein specificity and other desirable features. One of ordinary skill in the art would have found it beneficial to have a 4-HT-triggered intein that could be triggered to splice upon exposure to 4-HT as an approach to control protein function in cells post-translationally.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624) as applied to claim 1 above, and further in view of Lennon et al. (“Inteins”, Current Biology, 2017, Volume 27, Issue 6, pp.R204-R206).
The teachings of von der Heyde are discussed above.
Regarding claim 18, von der Heyde does not teach wherein the intein is inserted at or replaces a cysteine within the glycosyl hydrolase.
However, Lennon teaches that inteins work by self-splicing using 3 overlapping mechanisms (p.R205, 1st column). Lennon teaches that for the most common mechanism, class 1, the precursor undergoes a four-step reaction; first a nucleophile (serine or cysteine) at the start of the intein attacks the preceding peptide bond, generating a (thio)ester linkage (p.R205, 1st column; Figure 1). Lennon teaches that second, a nucleophile at the first position of the C-extein (a cysteine, serine or threonine) attacks the (thio)ester resulting in a branched intermediate (p.R205, 1st column; Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to insert the intein at a cysteine within the xylanase taught by von der Heyde, because Lennon teaches that inteins use a nucleophile at the start of the intein such as cysteine to attack the preceding peptide bond and generate a (thio)ester bond. One of ordinary skill in the art would reasonably expect that inserting an intein at a position containing a cysteine would predictably result in an intein that can self-splice by using the cysteine as a nucleophile, because it was known in the art at the time of invention that the most common mechanism of intein’s self-splicing ability relies on using cysteins as nucleophiles to activate the splicing mechanism.
Claims 22 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624) as applied to claim 1 above, and further in view of Pemberton et al. (“Mechanisms of receptor-mediated nuclear import and nuclear export”, Traffic, 2005, Vol. 6, Issue 3, pp.187-198) and Mikaélian et al. (“The DNA-Binding Domain of Two bZIP Transcription Factors, the Epstein-Barr Virus Switch Gene Product EB1 and Jun, Is a Bipartite Nuclear Targeting Sequence”, Journal of Virology, 1993, Vol. 67, No.2, pp.734-742).
The teachings of von der Heyde are discussed above.
Regarding claims 22 and 24, von der Heyde does not teach glycosyl hydrolase fused to a nuclear localization signal (claim 22). von der Heyde does not teach glycosyl hydrolase fused to a nuclear export signal (claim 24).
However, Pemberton teaches that karyopherins mediate either nuclear import or nuclear export (p.187, 1st column last paragraph). Pemberton teaches that karyopherins that mediate import bind to their cargoes in the cytoplasm via recognition of the nuclear localization signal (NLS) (p.187, 2nd column top paragraph). Pemberton further teaches that karyopherins that mediate export bind cargo in the nucleus via recognition of a nuclear export signal (NES) (p.187, 2nd column top paragraph).
Mikaélian teaches nuclear targeting signals (NTS) EB1 and Jun (abstract). Mikaélian teaches that EB1 and c-Jun BRA and BRB, are sufficient to direct β-galactosidase to the nucleus and act as a bipartite NTS (p.735, 1st column, 1st full paragraph). Mikaélian further teaches that when the construct encoding amino acids 1 to 227 of EB1 fused to the β-galactosidase was introduced in HeLa cells, β-galactosidase localized exclusively to the nucleus (p.736, 2nd column – EB1 basic region is sufficient to direct β-galactosidase to the nucleus).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the xylanase taught by von der Heyde with either a nuclear localization signal or a nuclear export signal taught by Pemberton to create an NTS-enzyme fusion protein taught by Mikaélian, because Pemberton teaches that cargo import and export is mediated by the recognition of nuclear import signals and nuclear export signals. One of ordinary skill in the art would reasonably expect that fusing a xylanase enzyme to a nuclear import signal or a nuclear export signal would predicably result in the relocation of the xylanase to the desired compartment, because Mikaélian teaches fusing a nuclear transport signal to β-galactosidase localized the fusion exclusively to the nucleus, and it was known in the art at the time of invention that nuclear localization signals and nuclear export signals guide the relocation of proteins into the desired cellular compartment.
Claim 26 is rejected under 35 U.S.C. 103 as being unpatentable over von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624) as applied to claim 1 above, and further in view of Gefen et al. (“Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome”, PNAS, 2012, Vol. 109, No. 26, pp.10298-10303).
The teachings of von der Heyde are discussed above.
Regarding claim 26, von der Heyde does not teach wherein the glycosyl hydrolase is fused to a targeting molecule.
Gefen teaches cohesin-fused β-glucosidase (abstract). Gefen teaches a chimeric cohesin-fused β-glucosidase binds directly to the cellulosome through an unoccupied dockerin molecule of its major scaffoldin subunit, thus focusing the β-glucosidase activity at the immediate cite of cellobiose production by the cellulosomal enzymes (abstract). Gefen teaches localization of BglA-CohII onto the cellulosome focuses the cellobiose-degradation activity at the general site of cellobiose accumulation where the enzyme would be more effective in reducing the inhibitory action of the disaccharide product on the cellulosomal enzymes (p.10299, 2nd column – 2nd paragraph). Gefen teaches that improvement of enzymatic deconstruction of lignocellulosic biomass is an essential step for effective production of plant derived biofuels (p.10299, 2nd column, Discussion).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the cohesin-fused β-glucosidase taught by Gefen for the xylanase taught by von der Heyde, because Gefen teaches that the cohesin-fused β-glucosidase taught by Gefen binds directly to the cellulosome through an unoccupied dockerin molecule of its major scaffoldin subunit. One of ordinary skill in the art would have been motivated to fuse the enzyme to a targeting molecule because Gefen teaches that β-glucosidase activity is focused at the immediate site of cellobiose production by the cellulosomal enzymes, making it more effective.
Claim 33 is rejected under 35 U.S.C. 103 as being unpatentable over Metcalf et al. (US 2016/0030528 A1, published on Feb. 4, 2016) in view of von der Heyde et al. (“A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein”, Applied Microbiology and Biotechnology, 2015, Vol. 99, pp.7613-7624).
Regarding claim 33, Metcalf teaches Aciduliprofundum boonei glycosyl hydrolase 25 (GH25) muramidase exhibits antibacterial activity against several distinct bacterial families (abstract). Metcalf teaches a pharmaceutical composition comprising an Aciduliprofundum boonei glycosyl hydrolase 25 muramidase domain disposed in a pharmaceutically acceptable diluent, carrier or excipient (description [0009]). Metcalf further teaches pharmaceutical formulations including classical pharmaceutical preparations [0078]; parenteral or intraperitoneal administration [0079]; and pharmaceutical forms suitable for injectable use and sterile injectable solutions [0080]-[0081]. Metcalf further teaches that the present compositions are useful in the treatment of infectious diseases, and in combined therapy against an infectious disease including antibiotics (description [0086], [0092]).
Metcalf does not teach a glycosyl hydrolase comprising an intein.
von der Heyde teaches a P. torridus intein inserted before amino acid residue Thr75 of a hemicellulase reporter enzyme from Pseudoalteromonas arctica (abstract). von der Heyde teaches intein-xylanase constructs (p.7621, Fig. 5A). As evidenced by Puchart, xylanases are glycoside hydrolases, and are part of the glycoside hydrolase families 10 and 11, and new family GH30 (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the glycosyl hydrolase 25 taught by Metcalf to fuse it to an intein, as taught by von der Heyde, in a pharmaceutical composition taught by Metcalf, because Metcalf teaches that Aciduliprofundum boonei glycosyl hydrolase 25 muramidase has antibacterial activity against several distinct bacterial families. One of ordinary skill in the art would have been motivated to create a pharmaceutical composition of a glycosyl hydrolase comprising an intein to control the activity of the glycosyl hydrolase after delivery. One of ordinary skill in the art would have found it beneficial to select a glycosyl hydrolase that had antibacterial activity against bacteria known to cause infectious disease for a pharmaceutical composition.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/DEEPA MISHRA/Examiner, Art Unit 1657
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