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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on October 6, 2025 has been entered.
Application Status and Withdrawn Rejections
Applicant’s amendments filed October 6, 2025, amending claim 1, 3 and 16, and cancelling claims 4, 6 and 12 is acknowledged. Claims 1-3, 5, 7-8 and 10-11, and 13-16 are pending and under examination.
The amendment to claim 3 overcomes the rejection under §112(d) of record. The cancelation of claims 4, 6 and 12 renders the rejections of those claims over Price and Dalia in view of Wang/Kidane or Westbrook moot. The rejection of claims 1-3, 5, 7-8 and 10-11, and 13-16 over Price in view of Dalia are withdrawn and in favor of rejections including additional references to address Applicant’s arguments and provide additional evidence for predictability of increasing homology arm length of donor sequences for integration in bacterial genomes.
Any other rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 13 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 13 recites “… when compared to the frequency of integration of said gene in a control method…” Claim 13 is indefinite because “said gene” lacks clear antecedent basis. Claim 1, from which claim 13 depends does not recite a “gene”. Although donor sequences can comprise genes, there is no limitation in claim 1 or previously recited in claim 13 for the donor DNA sequence to be or comprise a gene. Therefore, it is not clear what “said gene” is referring to.
To remedy the indefiniteness, it is suggested that claim 13 recite “…when compared to the frequency of integration of said donor sequence in a control method…”
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.
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.
Claims 1-3, 5, 7-8, 10-11 and 13-15 are rejected under 35 U.S.C. 103 as being unpatentable over Price (Price et al., PLoS ONE (2019), 14(1): e0210121, published January 7, 2019; of record), as evidenced by Genbank (Bacillus subtilis subsp. subtilis str. 168 complete genome, Accession NC_000964, https://www.ncbi.nlm.nih.gov/nuccore/255767013, available at least as early as September 18, 2018 [retrieved January 31, 2025]; of record), and in view of Dalia (US 20170051311 A1; of record) and Brigulla (Brigulla and Wackernagel, Appl Microbiol Biotechnol (2010), 86: 1027-1041) . This is a new rejection.
Regarding claims 1-3, Price teaches methods of genome editing in Bacillus subtilis (i.e., a Bacillus sp. cell) using CRISPR-Cas systems (Abstract). Price teaches co-transforming (i.e., introducing simultaneously) B. subtilis cells with plasmid pBAC0041 (i.e., a circular recombinant DNA construct) and a linear OE-PCR DNA construct (¶ spanning pages 4-5; Abstract). Regarding the circular recombinant DNA construct, Price teaches that the pBAC0041 plasmid encodes an sgRNA (i.e., a guide RNA) and a Cas9 endonuclease under the control of the Pgrac promoter (¶ spanning pages 4-5; Table 1). Price teaches that the Pgrac promoter has a basal level of expression sufficient for Cas9 expression (page 5, ¶1), and is therefore is a constitutive promoter operably linked to the nucleotide sequence encoding Cas9. Price teaches the Cas9 endonuclease creates a double strand break in the genomic site targeted by the sgRNA (spanning pages 4-5). Regarding the linear DNA construct, Price teaches the dDNA was constructed by overlap extension PCR (OE-PCR) to produce linear donor DNA (¶ spanning pages 4-5; Abstract). Price teaches the dDNA comprised sequences to introduce stop codons into the amyE locus (i.e., a DNA donor sequence comprising a polynucleotide, a terminator sequence, a heterologous sequence, a transgenic nucleic acid sequence) flanked on each side by sequences homologous to the amyE locus (i.e., an upstream homology arm (HR1) and a downstream homology arm (HR2)) (¶ spanning pages 4-5; see Fig 2B for pictorial representation at a different locus). Price teaches that the homologous sequences on each side of edits are approximately 1000 bp long (Fig 2B, scale bar). Price teaches the desired edits of the dDNA are integrated into the genome of the Bacillus cell (Fig 1; page 6, ¶4). Price teaches the method of gene editing is “markerless” (Abstract). Additionally, based on the disclosed methods of amyE and aprE locus editing in Price, it is evident that no selection marker was integrated into the genome of the Bacillus cell (page 4, ¶5 through page 5, ¶4; Fig 2). Regarding the length of the homology arms in the dDNA to edit the amyE locus, Price teaches the plasmid and OE-PCR donor were transformed into B. subtilis strain 168 (page 5, ¶1). Price teaches that the OE-PCR product was prepared using primers oMAP0121/0122/0128/0129 when the Cas9/sgRNA pBAC0041 was used (¶ spanning pages 4-5). Price teaches the sequences of primers oMAP0121, oMAP0122, oMAP0128, and oMAP0129 (Table S1).
Price is silent on the precise length of the homology arms in the dDNA used to edit the amyE locus in the B. subtilis strain 168.
Genbank teaches the genome sequence of B. subtilis strain 168, of which bases 326601-329600 are provided in the attached reference. Genbank teaches the amyE locus is from positions 1018-2997 in the shown range (page 3). As annotated in the Genbank reference primer, oMAP0121 is identical to positions 21-44 (page 4) and primer oMAP0122 is the reverse complement of positions 2139-2158 (pages 4). Comparing the sequences of primers oMAP0128 and oMAP0129 to the Genbank sequence, it is evident that the primers would produce an “AA” insertion at position 1043 to produce the stop codon “TAA” (page 3, arrow). The inserted “AA” is encompassed by the claimed “DNA donor sequence”. The upstream homology arm in the OE-PCR product spans from position 21-1043, which is 1023 nucleotides. The downstream homology arm in the OE-PCR product spans from position 1043-2158, which is 1116 nucleotides. Therefore, in Price’s method of integrating a donor DNA sequence into the amyE gene in the genome of B. subtilis, the donor DNA construct inherently comprised a 2-nt DNA donor sequence and upstream and downstream homology arms of 1023 and 1116 nucleotides in length.
Price does not teach upstream and downstream homology arms are 3000-6000 nucleotides in length.
Dalia teaches multiplex genome editing methods in microbial systems that are non-model organisms ([0006]). Dalia teaches one such microbial organism is Bacillus bacteria ([0013]). Dalia teaches contacting cells with nucleic acids sequence that comprise at least one homology arm that is homologous to a bacterial genome ([0095]; Figure 6A). Dalia teaches the homology arms can be a length in the range of 1000-4000 bases or less than 5 kb ([0095]). Dalia demonstrates an increased transformation and co-transformation frequency (i.e., integration) in the bacteria V. cholera when homology arms are lengthened from 1kb to 3 kb (Figure 1B, D-E; [0022]-[0024]).
Brigulla reviews the state of the art for foreign DNA acquisition in bacteria (Abstract). Brigulla teaches when foreign DNA that is flanked by two homologous sequences, two homologous recombination (HR) events can effectively integrate the foreign DNA in the bacterial genome (page 1030, ¶3). Brigulla teaches “In natural transformation with homology-embedded foreign DNA, quantitative experiments revealed that homologous flanks of several 100 nucleotides suffice for integration and that longer flanks (up to 10 kbp or more) increase the integration frequencies by up to a thousand-fold.” (page 1030, ¶3).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have increased the length of the homology arms of the donor DNA of Price from just over 1000 bp to 3000 bp and up to 5000 bp in Price’s method of integrating a donor DNA in a Bacillus cell. It would have amounted to lengthening a known DNA molecule by known means to yield predictable results. The skilled artisan would have predicted that the homology arms of Price’s dDNA could be lengthened because Dalia demonstrates longer homology arms of dDNA, which can be produced by the oePCR method in Price using different primer sequences. The skilled artisan would have been motivated to do so because both Dalia and Brigulla teach that using longer homology arms increases the efficiency of foreign DNA integration.
Regarding claim 5, Price teaches the donor DNA is a PCR product (i.e., a double strand DNA), as indicated above for claim 1.
Regarding claim 7, Price teaches growing the progeny from the transformed cells as evidenced by the ability to count colony-forming units (i.e., the colony comprises progeny from the transformed/edited cells) (Fig 1). Price teaches selecting progeny that possess the desired edit integrated into the genome by observing the presence or absence of the halo around the colonies on the transformation plates (Fig 1, legend).
Regarding claim 8, Price teaches that plasmid pBAC0041 was produced by cloning the Cas9 genes and sgRNA genes into pHT01, which comprises the bla gene (i.e., an ampicillin-resistance selection marker that is not integrated into the genome). Price teaches the method is “markerless” and there is no evidence that the ampicillin gene is integrated into the cell genome.
Regarding claim 10, Price also teaches introducing donor DNA at the aprE locus using a Cas9/sgRNA plasmid and linear donor DNA (Fig 2). Price teaches introducing two nucleotide changes in two sequential steps (Fig 2B-C; page 6, ¶5-6). Price teaches that before the second round of gene editing, there was a “curing process” (page 6, ¶6) which involved the removal of the Cas9/sgRNA plasmid due to lack of selection of the plasmid (page 5, ¶2).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have additionally removed the Cas9/sgRNA plasmid and linear PCR product from the amyE-edited cells. It would have amounted to applying a known method for modifying multiple sites through sequential rounds of editing to the amyE editing method of Price. The skilled artisan would have predicted that the Cas9/sgRNA plasmid could be removed after editing the amyE locus because Price demonstrates such a method after editing another genomic locus in the B. subtilis cells. Additionally, it would be expected that any non-integrated donor DNA would have been removed because the PCR product lacks an origin of replication and a selection marker. The skilled artisan would have been motivated to do so in order to prevent additional off-target editing by the amyE-targeted sgRNA or allow a subsequent round of editing in the amyE-edited cell.
Regarding claim 11, Price teaches the method is editing the endogenous amyE chromosomal locus (Abstract) as indicated for claim 1.
Regarding claim 13, Dalia teaches that the efficiency of transformants increases at least 10-fold when the homology arms were increased from 1 kb to 3 kb.
Regarding claim 14, Price teaches amyE editing using the claimed donor DNA in Bacillus subtilis cells (Abstract), as indicated above for claim 1.
Regarding claim 15, Price teaches co-transforming naturally competent B. subtilis 168 with the Cas9/sgRNA-encoding plasmid and the dDNA OE-PCR product (i.e., introduced by natural competence) (¶ spanning pages 4-5).
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Price (Price et al., PLoS ONE (2019), 14(1): e0210121, published January 7, 2019; of record), evidenced by Genbank (Bacillus subtilis subsp. subtilis str. 168 complete genome, Accession NC_000964, https://www.ncbi.nlm.nih.gov/nuccore/255767013, available at least as early as September 18, 2018 [retrieved January 31, 2025]; of record), in view of Dalia (US 20170051311 A1), Brigulla (Brigulla and Wackernagel, Appl Microbiol Biotechnol (2010), 86: 1027-1041), Berka (US 20190024123 A1, published January 24, 2019; of record) and Schumann (Wolfgang Schumann, “Production of Recombinant Proteins in Bacillus subtilis.” Advances in Applied Microbiology (2007), Vol 62: 137-189; of record). This is a new rejection.
The teachings of Price, Genbank, and Dalia are recited above in paragraphs 13, 15, 17, and 18 and incorporated here.
Briefly, Price as evidenced by Genbank, teaches methods of genome editing in Bacillus subtilis using CRISPR-Cas systems (Abstract). Price teaches co-transforming B. subtilis cells with a plasmid encoding a guide RNA and a Cas9 endonuclease under the control of the constitutive Pgrac promoter (¶ spanning pages 4-5; Table 1; Abstract) and a linear DNA construct comprising ~ 1000 bp homology arms flanking a stop codon that is to be inserted into the amyE locus (¶ spanning pages 4-5; Fig 1 and 2B). Price teaches the method of gene editing is “markerless” (Abstract). Dalia teaches multiplex genome editing methods in microbial systems that are non-model organisms ([0006]), including Bacillus bacteria ([0013]). Dalia teaches homology arms can be a length in the range of 1000-4000 bases or less than 5 kb ([0095]). Dalia demonstrates an increased foreign DNA integration in the bacteria V. cholera when homology arms are lengthened from 1kb to 3 kb on a linear donor DNA (Figure 1B, D-E; [0022]-[0024]). Brigulla reviews the state of the art for foreign DNA acquisition in bacteria and teaches it is generally expected that integration of larger inserts increases with longer flanking sequences (page 1030, ¶3).
Price, Dalia and Brigulla do not teach including multiple copies of a single gene of interest in the donor DNA sequence.
Westbrook teaches methods of integrating donor DNA in Bacillus subtilis cells using Cas9 and guide RNAs (Abstract). Westbrook teaches methods for integrating entire genes and operons into a targeted site in the Bacillus genome (Abstract, Fig 7C, page 4886, ¶2). Westbrook teaches the editing template (i.e., donor DNA) for introducing an entire HA operon into the amyE locus was linearized plasmid pAW020-2 (¶ spanning pages 4882-4883). Westbrook teaches that integrating the HA operon in to B. subtilis allows for high-level production of a high-value therapeutic polymer (page 4886, ¶2).
Berka teaches using Cas9/sgRNA-mediated gene editing in Bacillus host cells to integrate a donor DNA at a target locus (Abstract, Fig 2). Berka teaches that recombinant protein production can include multiples copies of a gene encoding the substance ([0034]).
Schumann teaches methods and considerations for using B. subtilis for recombinant protein production (Title; ¶ spanning pages 138-139). Schumann teaches integrative vectors allow ectopic insertion of cloned genes into sites of the chromosome to avoid the problem of plasmid instability (page 146, ¶2). Schumann teaches that a common integration site is the amyE locus (page 146, ¶3). Schumann teaches that one issue with transgene integration is lower transgene copy number, but that can be solved by increasing the copy number of the recombinant gene within the chromosome (page 147, ¶2). Schumann teaches that gene amplification in the chromosome is desirable for enhanced expression of recombinant genes (page 148, ¶1).
The obviousness of increasing the lengths of the homology arms of the donor DNA to 3000-5000 nucleotides in Price is recited above as for claim 1. It also would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have integrated two copies of the HA-operon into the amyE locus using a linearized vector as taught in Westbrook, in the obvious method of amyE-editing using a donor DNA comprising 3-5 kb long homology arms. It would have amounted to the simple substitution of one editing sequence for another and the simple combination of elements by known means to yield predictable results. The skilled artisan would have been motivated to replace the two-nucleotide sequence that codes for a stop codon for the HA-operon for the purpose of using B. subtilis to produce high-value therapeutic polymers as indicated in Westbrook. There was a reasonable expectation of success of including the HA-operon-coding sequence between the longer homology arms because Westbrook and Brigulla teaches longer homology arms results in higher frequencies of editing of the amyE locus. The skilled artisan would have predicted that two copies of the HA-operon could be included in a linearized vector because Berka teaches that isolated polynucleotides can have multiple copies of a gene. One would have been motivated to do so because Schumann teaches that having multiple copies of a gene integrated into the B. subtilis genome increases expression of the recombinant proteins and avoids plasmid instability.
Response to Arguments - §103
Applicant summarizes the obviousness rejection over Price in view of Dalia (Remarks, page 6). Applicant argues that Dalia provides no experimental data, examples, or rationale specific to Bacillus species as Dalia’s experimental work is conducted in Vibrio cholera (page 7, ¶1-2). Applicant compares the transformation and recombination machinery between Vibrio and Bacillus to support their conclusion that the skilled artisan would not have been motivated by the evidence in Dalia to increase the length of the homology arms in the method of Price. Applicant provides references that allegedly teach 1) the structural machinery of competence (i.e., DNA uptake) is different; 2) the regulatory machinery is different; and 3) DNA substrate and processing is different (Remarks, pages 7-9). These arguments have been fully considered but are not persuasive for the following reasons.
First it should be noted that MPEP 2143.02 teaches that obviousness does not require absolute predictability, but only a reasonable expectation of success is required. Because Price teaches all the limitations of the claim except for the length of the homology arms, Dalia does not need to demonstrate homologous recombination (HR) methods in Bacillus. The question at hand is whether the skilled artisan with knowledge in the art about bacterial DNA competence, transformation, and HR would have been motivated by Dalia to increase the length of the homology arms in Price’s Cas9/guide RNA-mediated donor sequence integration method. Examiner agrees that Dalia only presents increased integration efficiency in Vibrio. However, as taught in Brigulla, there was a general understanding in the art that increasing the length of the homology arms increases double-crossover HR in many bacterial species. For instance, Simpson teaches in Acinetobacter baylyi that when the ratio of homology arm length to insert length increases, integration efficiency increases (Simpson et al., Environ. Biosafety Res. (2007), 6: 55-69; Fig 2). Piven found that as the flanking homology regions were increased from 2 kb to 3kb, the rate of HR increased in cyanobacteria by 10-fold (US 20140178958 A1, [0541]; FIG 104). Thus, the general understanding in the art of HR-mediated bacterial gene integration was 1) longer homology arms generally correlate with higher integration efficiencies and 2) longer homology arms allow integration of larger foreign DNA inserts.
Applicant’s cited teachings of the differences in Vibrio and Bacillus competence machinery and regulation are not sufficient to dissuade the skilled artisan to ignore the findings the Dalia. Although the bacterial species use somewhat different machinery to take up DNA from the environment, the overall process is quite similar. Blokesche teaches that the general core uptake machinery is conserved among naturally competent species except for the expected differences between gram+ and gram- species and points out that both species import DNA as single stranded DNA (Current Biology (2016), R1119-R1136; pages R1127-R1128). Additionally, the DNA important machinery of gram+ and gram- bacteria illustrated in Figures 1 and 2, respectively, in the Niu et al. references cited by authors, are strikingly similar (Frontiers in Biology (2025), 16: 1578813). Blokesche, Niu, Dalia and Brigulla each provide evidence that it was well-known in the art how to induce natural competence in both Bacillus and Vibrio. Thus, there is no debate that both species are capable of importing large pieces of DNA that comprise both foreign sequences and homologous sequences. Additionally, both Blokesche and Niu teach that the competence-triggering factors of both Bacillus and Vibrio are known (Blokesche page R1127, Table 1; Niu, Section 3.1). The fact that their machinery may be a little different to account for their different cell wall components and the factors that induce expression of their machinery would not dissuade the skilled artisan since the means to induce import large DNA in both species are known. The skilled artisan need only grow the bacteria according to known natural competence methods to induce large DNA uptake.
Applicant’s citations of the differences in homologous recombination in Vibrio and Bacillus are not sufficient to dissuade the skilled artisan to ignore the findings the Dalia. First, the fact that HR in Vibrio is more efficient with plasmids is not pertinent to whether it was predictable that Dalia’s experimental data could be applied to HR in Bacillus because Dalia’s HR data used a linear donor DNA for HR. Niu, which was cited by Applicant, states “In gram-negative bacteria, the process of DNA homologous recombination is similar to that in Gram-positive bacteria” (Section 2.2.2). This direct statement supports the conclusion that the skilled artisan would reasonably expect that means for increasing linear DNA HR frequencies in the gram-negative bacteria Vibrio would also result in increased linear DNA HR rates in the gram-positive bacteria, Bacillus subtilis.
Applicant argues that “a plasmid system optimized for Vibrio may fail in Bacillus due to differences in replication origin compatibility (page 9, ¶2). This argument has been fully considered but is not persuasive because it does not address the merits of the rejection. Price already teaches all of the components of the claimed method except for the length of the homology arms. The only teaching relied upon in Dalia is that longer homology arms in linear DNA donor sequences increased integration efficiency. Thus, the “plasmid system” of Dalia is not used in the method rendered obvious in the rejection for claim 1.
Applicant argues that for the reasons provided for claim 1 (i.e., differences between Vibrio and Bacillus) the teachings do not provide an expectation of success in Bacillus (Remarks, page 11, ¶1-4). This argument has been fully considered but is not persuasive because as indicated in the previous paragraphs, it was known in the art that Bacillus could import large linear DNA molecules and the homologous recombination mechanisms between gram- and gram+ bacteria were generally similar.
Applicant argues that each reference only teaches a subset of the claimed elements and there was no motivated in the art to combine them in the manner claimed (Remarks, page 11, ¶6). This argument has been fully considered but is not persuasive because obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Price teaches (i) a plasmid-based Cas9/sgRNA system, (ii) a linear donor DNA construct, and (iv) markerless integration in Bacillus. Dalia teaches (ii and iii) a linear donor DNA construct with homology arms of 3000+ nucleotides and a motivation to increase homology arm length from 1000 [Wingdings font/0xE0] 3000 nucleotides (i.e., increased integration efficiency). Westbrook, Berka, and Schumann teach integrating gene operons and multiple copies of gene operons and provide the motivation to use Cas9/sgRNA to integrate multiple copies of genes. Thus, each of the elements of claim 16 are taught in the prior art and motivation to incorporate each of the elements into Price’s method of Cas9/gRNA-mediated engineering of Bacillus is provided in the rejection above.
Applicant attests to the advantages of the claimed method and appears to argue that the claimed method provides surprising results of efficient markerless integration of multiple copies of a gene into the Bacillus genome (Remarks, page 11, last ¶ to page 12, ¶2). This argument has been fully considered but is not persuasive because they are merely arguments of counsel. MPEP 716.01(c) makes clear that arguments of counsel cannot take the place of evidence in the record.
Conclusion
No claims are allowable.
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/CATHERINE KONOPKA/Examiner, Art Unit 1635