DETAILED ACTION
Notice of 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 .
Status of Application
Claims 1-15 are pending and subject to examination on the merits.
Priority
The instant application is a 371 of PCT/US2023/072196 filed 15 August 2023 which claims benefit of US provisional application 63/371,616.
Information Disclosure Statement
The information disclosure statement (IDS) submitted 23 February 2025 has been considered by the examiner. See initialed and signed PTO/SB/08.
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.
Claim(s) 1-4 and 6-15 are rejected under 35 U.S.C. 103 as being unpatentable over Budinoff et al. (WO 2022178214 – cited herein) in view of Kimmenade et al. (US 2011/0104786 – cited on herein).
Budinoff et al. teach phosphates, such as apyrases, can be utilized in the medical industry to treat gut inflammation (See paragraph 0007). Specifically, they teach nucleic acid SEQ ID NO: 28 in Example 4 has 99% sequence identity to instant nucleic acid SEQ ID NO: 9 (Supplemental Content, 20260918_104115_us-19-103-587a-9.rng file, Result #3) and 99.7% sequence identity to instant nucleic acid SEQ ID NO: 37 (See Supplemental Content, 20260918_104115_us-19-103-587a-37.rng file, Result #3), and encodes phosphatase/apyrase enzyme SEQ ID NO: 9 (CRC22110) - (See Table 4, Example 4; Claim 3), wherein the Budinoff et al. amino acid sequence SEQ ID NO: 9 has 99.3% sequence identity to instant amino acid sequence SEQ ID NO: 10 (See Supplemental Content, 20260918_104141_us-19-103-587a-10.rag file, Result #2); 100% sequence identity to instant amino acid sequence SEQ ID NO: 25 (See Supplemental Content, 20260918_104141_us-19-103-587a-25.rag file, Result #1), 100% sequence identity to instant amino acid sequence SEQ ID NO: 26 (See Supplemental Content, 00260918_104141_us-19-103-587a-26.rag file, Result #7) and 100% sequence identity to instant amino acid sequence SEQ ID NO: 27 (See Supplemental Content, 20260918_104141_us-19-103-587a-27.rag file, Result #3). Said enzyme is cloned into and expressed in Bacillus subtilis host cells and then purified for enzymatic characterization to ascertain which enzymes have the greatest enzymatic activity, wherein CRC22110/SEQ ID NO: 9 has significant enzymatic activity (See Tables 5 and 6), and wherein said enzyme is secreted from the host cell (paragraph 0154, Ex. 4). As noted, the enzymes are to be utilized in the treatment and prevention of gut inflammation at thus are industrially relevant enzymes (See Abstract and Summary). It is noted, said sequence has 14 cysteine residues and thus inherently will have one to seven disulfide bonds and is enzymatically active when produced in Bacillus subtilis host cells.
Budinoff et al., however, are silent with regard to which B. subtilis host cell was utilized to express said enzyme(s) and that they had at least one to nine endogenous protease genes deleted, such as at least one of aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and/or nprB (claims 1, 4, 6).
Kimmenade et al. teach strains and methods of producing industrially useful enzymes of interest in B. subtilis host cells starting from strain 168 (See paragraph 0003-0006, Example 5), wherein said host cells are modified to have reduced endogenous proteases. Specifically, strain BG6000 is taught to have the following modifications: (ΔaprE, ΔnprE, Δepr, ΔispA, Δbpf, Δvpr, oppA, ΔspoIIE, degUHy32, ΔamyE::(xylR,pxylA-comK)). It is further taught that said BG6000 strain further lacks WprA and/or mpr (See paragraphs 0007, 0009); wherein bpf and the instantly recited bpr are one and the same, e.g. a Bacillopeptidease F protease – see paragraph 0341. The additional genes that are deleted or disrupted or attenuated in the strain are permitted given the comprising language in the instant claims. Said introduction of the desired enzyme takes place on extrachromosomal plasmids/vectors or are integrated into chromosomes of the host cell (See paragraphs 0039-0041). In paragraph 0005, it is stated when producing enzymes of interest in host cells (such as Bacillus) there is need to produce said enzymes in a host strain lacking detrimental endogenous protease activity.
Therefore, it would have been obvious to one skilled in the art prior to the effective filing date of the claimed invention to express the apyrase gene of SEQ ID NO: 28, which encodes the phosphatase of SEQ ID NO: 9 (CRC22110) as taught by Budninoff et al. in the B. subtilis protease deficient expression host cell as B. subtilis taught by van Kimmenade et al. (ΔaprE, ΔnprE, Δepr, ΔispA, Δbpf, Δvpr, oppA, ΔspoIIE, degUHy32, ΔamyE::(xylR,pxylA-comK) and further deletes wprA or mpr) in order to overexpress said phosphatase/apyrase because Budinoff et al. teach how important this enzyme is for treatment of gut inflammation diseases and van Kimmenade et al. teach their strains with endogenous proteases that are deleted are useful for making industrially desirable enzymes. This would be motivation in and of itself because the phosphatase/apyrase of Budinoff et al. is an important enzyme in the medical field and industry. One skilled in the art would have a reasonable expectation of success in expressing the phosphatase/apyrase enzyme as taught by Budinoff et al. which is expressed in an unidentified B. subtiltis host cell, and to instead express it in the protease deficient B. subtiltis host cell of van Kimmenade et al. (e.g. BG6000 - (ΔaprE, ΔnprE, Δepr, ΔispA, Δbpf, Δvpr, oppA, ΔspoIIE, degUHy32, ΔamyE::(xylR,pxylA-comK)) additionally with wprA and/or mpr deleted, because Budinoff et al. already exemplify the success of expressing said enzyme in B. subtilis and van Kimmenade et al. teach it is useful and helpful to delete “detrimental endogenous proteases” (See paragraph 0005) when using B. subtilis as the host cell for producing desired industrially desirable enzymes.
Claim(s) 1-15 are rejected under 35 U.S.C. 103 as being unpatentable over Budinoff et al. (WO 2022178214 – cited herein) in view of Jeong et al. (Microbiology Resource Announcements, 2018 – cited herein) and Fox et al. (US 20080090745 – cited herein).
Budinoff et al. teach phosphates, such as apyrases, can be utilized in the medical industry to treat gut inflammation (See paragraph 0007). Specifically, they teach nucleic acid SEQ ID NO: 28 in Example 4 has 99% sequence identity to instant nucleic acid SEQ ID NO: 9 (Supplemental Content, 20260918_104115_us-19-103-587a-9.rng file, Result #3) and 99.7% sequence identity to instant nucleic acid SEQ ID NO: 37 (See Supplemental Content, 20260918_104115_us-19-103-587a-37.rng file, Result #3), and encodes phosphatase/apyrase enzyme SEQ ID NO: 9 (CRC22110) - (See Table 4, Example 4; Claim 3), wherein the Budinoff et al. amino acid sequence SEQ ID NO: 9 has 99.3% sequence identity to instant amino acid sequence SEQ ID NO: 10 (See Supplemental Content, 20260918_104141_us-19-103-587a-10.rag file, Result #2); 100% sequence identity to instant amino acid sequence SEQ ID NO: 25 (See Supplemental Content, 20260918_104141_us-19-103-587a-25.rag file, Result #1), 100% sequence identity to instant amino acid sequence SEQ ID NO: 26 (See Supplemental Content, 00260918_104141_us-19-103-587a-26.rag file, Result #7) and 100% sequence identity to instant amino acid sequence SEQ ID NO: 27 (See Supplemental Content, 20260918_104141_us-19-103-587a-27.rag file, Result #3). Said enzyme is cloned into and expressed in Bacillus subtilis host cells and then purified for enzymatic characterization to ascertain which enzymes have the greatest enzymatic activity, wherein CRC22110/SEQ ID NO: 9 has significant enzymatic activity (See Tables 5 and 6), and wherein said enzyme is secreted from the host cell (paragraph 0154, Ex. 4). As noted, the enzymes are to be utilized in the treatment and prevention of gut inflammation at thus are industrially relevant enzymes (See Abstract and Summary). It is noted, said sequence has 14 cysteine residues and thus inherently will have one to seven disulfide bonds and is enzymatically active when produced in Bacillus subtilis host cells.
Budinoff et al., however, are silent with regard to which B. subtilis host cell was utilized to express said enzyme(s) and that they had at least one to nine endogenous protease genes deleted, such as at least one of aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and/or nprB (claims 1, 4-6).
Jeong et al. teach that Bacillus subtilis WB800N is a genetically engineered variant of B. subtilis 168, such that all extracellular proteases are disrupted, which enables WB800N to be widely used for the expression of secretory proteins. They teach the 4.2-Mb complete genome sequence of WB800N and present all of the disrupted gene structures, and teach that the following nine proteases are inactivated or deleted: aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and nprB.
Fox et al. teach inactivation of nine endogenous proteases in B. subtilis host cells for production of a heterologous enzyme, wherein the nine proteases are aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and nprB (See Claim 1, paragraph 0060), wherein inactivation includes deletion (See paragraph 0042).
Therefore, it would have been obvious to one skilled in the art prior to the effective filing date of the claimed invention to express the apyrase gene of SEQ ID NO: 28, which encodes the phosphatase of SEQ ID NO: 9 (CRC22110) as taught by Budninoff et al. in the B. subtilis protease deficient expression host cell as B. subtilis WB800N as taught by Jeong et al. or the B. subtilis host cell of Fox et al., which has nine endogenous proteases inactivated and/or deleted (aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and nprB) in order to overexpress said phosphatase because Budinoff et al. teach how important this enzyme is for treatment of gut inflammation diseases and both Jeong et al. and Fox et al. teach their strains with endogenous proteases that are deleted are useful for making industrially desirable enzymes. This would be motivation in and of itself because the phosphate/apyrases of Budinoff et al. is an important enzyme in the medical field and industry. One skilled in the art would have a reasonable expectation of success in utilizing the phosphatase/apyrase enzyme as taught by Budinoff et al. which is expressed in a B. subtiltis host cell, and to instead express it in the protease deficient B. subtiltis host cell of Jeong et al. and/or Fox et al. which have nine endogenous proteases inactivated and/or deleted (aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and/or nprB), because Budinoff et al. already exemplify the success of expressing said enzyme in B. subtilis and Jeong et al. teach WB800N is widely used for the expression of secretory proteins and Fox et al. teach inactivation and/or deletion of the nine endogenous proteases successfully produces industrially useful enzymes.
Conclusion
No claim is allowed.
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/SUZANNE M NOAKES/Primary Examiner, Art Unit 1656 21 September 2026
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