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 .
Election/Restrictions
Applicant's election with traverse of Group I, claims 7-20, in the reply filed on 9/16/2025 is acknowledged. The traversal is on the ground(s) that Group I and II share a special technical feature and should be examined together. Specifically, Applicant argues that Gill, et al. describes a bacteriophage (Accession No. YP_002922742.1) comprising a gene encoding lysozyme fused to a DarB-like antirestriction protein, not DarB per se, so there is unity of invention. This is not found persuasive because, as demonstrated below by the rejection under 35 U.S.C. §103, the shared technical feature of a bacteriophage comprising a gene encoding lysozyme fused to DarB is not a special technical feature. It cannot be considered inventive in view of its obviousness discussed below, al said discussions being incorporated here.
Claims 21-26 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 9/16/2025.
Claims 7-20 are under examination on the merits.
Priority
This application is a 371 of PCT/JP2020/029188 filed on 7/30/2020. The U.S. effective filing date of all claims under examination is set at 07/30/2020.
Information Disclosure Statement
The Information Disclosure Statement (IDS) submitted on 1/30/2023 is in compliance with 37 CFR 1.97. Accordingly, the IDS is being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Interpretation
DarB was first described as a P1 phage protein encoded by darB (defense against restriction), which provides protection against type I restriction systems, and is found in the phage head and protects any DNA packaged into a phage head from restriction (see, Iida, et al. Virology 157, 156-166, 1987).
The instant specification does not specifically define DarB, but says “the DarB of the bacteriophage of the invention is a rare DarB protein in which the two are fused. Due to the lysozyme fused-type DarB protein in which lysozyme is fused, the bacteriophage of the invention can avoid the restriction mechanism of the host bacterium, replicate the own genetic information efficiently in the host, lyse the host bacterium efficiently and grow efficiently (para. [0015]). Examples of such a fused gene include gp70 (YP_002922742.1) of Burkholderia virus BcepIL02, gp75 (NP_944303.1) of Burkholderia virus Bcep22, gp65 (YP_006589997.1) of Burkholderia virus DC1, the gene from the 44,008th to the 57,543rd residues (ORF66) of the base sequence of SEQ ID NO: 1 and the like. Of the genes encoding lysozyme fused DarB, the gene from the 44,008th to the 57,543rd residues (ORF66) of the base sequence of SEQ ID NO: 1 is preferable (Id.).
In Applicant’s response to the restriction requirement filed 8/28/2025, Applicant states that Gill, et al. describes a bacteriophage (Accession No. YP_002922742.1) comprising a gene encoding lysozyme fused to a DarB-like antirestriction protein, not DarB per se.
Thus, the examiner is interpreting the claim term “DarB” to represent a genus containing all known variants thereof, either natural or lab-made that use DarB as a name. However, the examples of such a fused gene above, based on Applicant’s arguments, are not within the scope of the lysozyme fused “DarB” currently claimed. Rather, the specification is merely describing similar fusions outside claim scope.
Claim Objections
Claim 12 is objected to because of the following informalities: the claim is missing an article at the start of the preamble. “The” should be added at the beginning of claim 12. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 12 (as presented) is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-ATA), first paragraph, as failing to comply with the enablement requirement. The claim contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The invention appears to disclose and employ a biological material, in the form of a bacteriophage “RKP180” (having deposit number NITE BP-03185; that infects Ralstonia solanacearum bacteria; see instant specification, [0002] and [0037], in particular). Since the biological materials are essential to the claimed invention they must be obtainable by a repeatable method set forth in the specification or otherwise readily available to the public. If the biological material is not so obtainable or available, the requirements of 35 U.S.C. §112 may be satisfied by a deposit of the biological material. The specification does not disclose a repeatable process to obtain the biological material and it is not apparent if the biological material is readily available to the public.
It is noted that applicant has deposited the biological material (instant specification, page no. 7, [0037], in particular), but there is no indication in the specification as to public availability. If the deposit is made under the Budapest Treaty, then an affidavit or declaration by applicant, or a statement by an attorney of record over his or her signature and registration number, stating that the specific biological material (in the instant case, bacteriophage RKP180 having deposit number NITE BP-03185) has been deposited under the Budapest Treaty and that the biological material will be irrevocably and without restriction or condition released to the public upon the issuance of a patent, would satisfy the deposit requirement made herein. If the deposit has not been made under the Budapest Treaty, then in order to certify that the deposit meets the criteria set forth in 37 C.F.R. §1.801-1.809, applicant may provide assurance of compliance by an affidavit or declaration, or by a statement by an attorney of record over his or her signature and registration number, showing that:
(a) during the pendency of this application, access to the invention will be afforded to the commissioner upon request;
(b) all restrictions upon availability to the public will be irrevocably removed upon granting of the patent;
(c) the deposit will be maintained in a public depository for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer;
(d) a test of the viability of the biological material at the time of deposit will be made (see 37 C.E.R. §1.807); and
(e) the deposit will be replaced if it should ever become inviable.
Applicant’s attention is directed to MPEP §2400 in general, and specifically to §2411.05, as well as to 37 C.F.R. §1.809(d), wherein it is set forth that “the specification shall contain the accession number for the deposit, the date of the deposit, the name and address of the depository, and a description of the deposited material sufficient to specifically identify it and to permit examination”. The specification should be amended to include this information; however, applicant is cautioned to avoid the entry of new matter into the specification by adding any other information. Appropriate correction is required.
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.
Claims 8, 10, 12, 14, and 16 are 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 8 recites the limitation "the above-mentioned lysozyme fused to DarB, and Rz/Rz1" in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 10 recites the limitation "the host recognition/binding" in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 10 is also indefinite because it is unclear what “binding” refers to. It appears to the examiner that a word is missing after “binding” that would clarify the subject of the “binding”.
Claim 10 is also indefinite because it recites the limitation “a gene encoding a tail fiber protein(s)” (emphasis added) on lines 1-2. It is unclear whether the claim requires i) a single tail fiber protein or ii) one or more tail fiber proteins, due to the wording of the claim. The presence of multiple interpretations renders the claim indefinite.
Claim 10 recites the limitation “having a receptor binding domain related to the host recognition/binding at the C-terminus” on lines 2-3. There are multiple interpretations for the phrase “related to”. For example, the relation could be phylogenetic or by sequence percent identity. Further, it is unclear what level of phylogenetic or sequence percent identity is sufficient to read on the claim.
Claim 12 recites the limitations “RKP180 (NITE BP-03185)”, which is an ambiguous recitation for a presumed biological deposit of a bacteriophage (as per disclosure of record; see specification, page 7, para. [0037], for instance) in parentheses, because it is unclear if the limitations presented in parentheses are part of the claimed invention that is bacteriophage “RKP180”, or just an exemplary recitation for said phage product. Thus the metes and bounds of the claimed invention does not appear to be properly defined. Appropriate correction is required.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 14 recites the broad recitation “double-stranded genome” on line 3, and the claim also recites “genomic DNA” on line 5, which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Claim 16 recites the limitation "the Ralstonia solanacearum" in line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 16 is also indefinite because it recites “the Ralstonia solanacearum is strain MAFF10726 [additional strains] and strain MAFF301558” (emphasis added). The claim appears to state that the Ralstonia solanacearum is multiple strains at once, which is unclear.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 7-16 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural product without significantly more.
“Determining that a claim falls within one of the four enumerated categories of patentable subject matter recited in 35 U.S.C. 101 (i.e., process, machine, manufacture, or composition of matter) [..] does not end the eligibility analysis, because claims directed to nothing more than [..] natural phenomena, and laws of nature are not eligible for patent protection.” MPEP §2106.04(I).
In the instant case, the claims are directed to a category of patentable subject matter (a composition) recited in 35 U.S.C. §101, because they recite “[a] bacteriophage comprising a gene encoding lysozyme fused to DarB” (claim 7). As discussed in the claim interpretation section above, claim 7 has been interpreted to encompass natural phages, including RKP180. The specification explains that “ORF66 of RKP180 encodes a fusion protein in which a lysozyme-like lytic enzyme and DarB are fused” (para. [0061]; Fig. 7). The specification also indicates that RKP180 is a natural phage isolated from soil (spec., Example 1).
This judicial exception is not integrated into a practical application because claims 7-16 and 18-20 do not recite additional elements that integrate the judicial exception into a practical application. “[M]ere recitation of a judicial exception does not mean that the claim is "directed to" that judicial exception under Step 2A Prong Two. Instead, under Prong Two, a claim that recites a judicial exception is not directed to that judicial exception, if the claim as a whole integrates the recited judicial exception into a practical application of that exception. Prong Two thus distinguishes claims that are ‘directed to’ the recited judicial exception from claims that are not "directed to" the recited judicial exception.” MPEP §2106.04(II)(A)(2).
The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the additional elements of the claims are either other genes or characteristics of the natural phage (claims 8-16).
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.
Claims 7, 10, 11, 13, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Gill, et al. (J Bacteriol. 2011 Oct;193(19):5300-13. doi: 10.1128/JB.05287-11. Epub 2011 Jul 29. PMID: 21804006; hereinafter referred to as “Gill”) in view of Fokine, et al. (J Biol Chem. 2008 Mar 14;283(11):7242-50. doi: 10.1074/jbc.M709398200. Epub 2007 Dec 26. PMID: 18160394; hereinafter referred to as “Fokine”), Iyer (J Bacteriol. 2017 Jul 11;199(15):e00245-17. doi: 10.1128/JB.00245-17. PMID: 28559295; hereinafter referred to as “Iyer”) and Kumar (Dissertation by Denish Kumar Piya, Interactions Between Host and Phage Encoded Factors Shape Phage Infection, Texas A&M University, December 2018; hereinafter referred to as “Kumar”).
The claimed invention encompasses a bacteriophage comprising a gene encoding lysozyme fused to DarB (claim 7). In one embodiment, the bacteriophage further comprises a gene encoding a tail fiber protein having a receptor binding domain related to host recognition/binding at the C-terminus (claim 10). In another embodiment, the bacteriophage comprises a linear genome or a circular permuted structure (claim 11). Alternatively, the bacteriophage is belonging to family Podoviridae of order Caudovirales of Group I (claim 13).
In another embodiment, bacteriophage is produced by modifying or mutating genomic DNA of a bacteriophage to contain a gene encoding lysozyme fused to DarB (claim 17).
The Prior Art
Gill discloses characterization of phages BcepIL02 and Bcep22, which are members of Podoviridae and nearly identical morphologically, bearing ~70 nm isometric heads and short, noncontractile tails (Abstract; p. 5302, col. 1). The genomes of both phages appear circularly permuted, and are 62,714 (66.2% G+C) and 63,882 (65.3% G+C) bases in length, respectively (p. 5302, col. 2, para. 2). BcepIL02 and Bcep22 encode strikingly large virion-associated proteins, gp70 (4,667 aa) and gp75 (4,602 aa), which appear to be functional equivalents of the 2,255 aa phage P1 DarB protein. (p. 5308, col. 2, para. 2). P1 DarB is required for protecting P1 DNA from restriction by the host type I restriction system, and may bind to or modify the cognate phage DNA during or shortly after its ejection into the host cell (Id.). Each of DarB, gp70, and gp75 is virion associated, contains a methyltransferase domain, and a DExH helicase domain (Id.). In addition to being considerably larger than P1 DarB, both gp75 and gp70 contain an N-terminal soluble lytic transglycosylase (SLT) domain that is absent from all other DarB-like proteins identified (p. 5308, col. 2, para. 3). Additionally, BcepIL02 and Bcep22 encode endolysins, detected as N-acetylmuramoylhydrolase lysozymes, of the same class as the soluble lysozyme found in phage T4 (p. 5308, col. 1, para. 4). Gill further discloses that most phages encode two spanin subunits, the functional equivalents of the co-liphage lambda Rz and Rz1 proteins (p. 5308, col. 1, para. 3). The Rz1 homologs of phages BcepIL02 and Bcep22, gp76 and gp81, respectively, belong to the embedded class of Rz1 genes, in that the entire Rz1-equivalent reading frame is embedded in the +1 reading frame of the Rz equivalent (Tables 1-2; p. 5308, col. 2, para. 1). Additionally, Gill discloses that BcepIL02 and Bcep22 encodes genes homologous to CsrA, orf49 and orf54, respectively (Table 1). Furthermore, phages BcepIL02 and Bcep22 contain multiple putative tail fiber protein orthologs (p. 5307, col. 1, para. 2), several of which contain receptor-binding domains in their C termini (p. 5306, col. 2, para. 1). Gill also discloses that phages Bcep22 and BcepIL02 contain closely related lysis cassettes with the canonical gene order of holin-endolysin-Rz/Rz1 (p. 5308, col. 1, para 3). However, Gill does not specifically teach a bacteriophage comprising a gene encoding lysozyme fused to DarB.
Fokine teaches that lytic transglycosylases are enzymes that act on peptidoglycan of bacterial cell walls, cleaving the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues (Abstract). Fokine further discloses that Pseudomonas bacteriophage φKZ gp144 is a lytic transglycosylase employed by the bacteriophage to facilitate release of the phage progeny (Abstract).
Iyer discloses that polyvalent proteins are involved in intergenomic biological conflicts of bacteriophages, and are transmitted by genomic parasites, such as phages, during the initial phase of infection along with DNA; polyvalent proteins are typified by the presence of multiple domains with disparate activities combined in the same protein (p. 1, para. 2, Importance). Iyer further discloses that DarA and DarB proteins bind the transferred phage DNA, protecting it against restriction systems (p. 3, para. 1). Iyer also discloses that DarB contains DNA methylase and helicase domains (pp. 12-13, bridging para.; p. 13, paras 2-3).
Kumar discloses phage-host interactions have resulted in the development of several defense and counter-defense strategies such as DNA restriction and antirestriction systems (Abstract). Type I restriction-modification systems present a barrier to foreign DNA, including phage, entering the bacterial cell, by cleaving inappropriately modified DNA in a sequence-specific manner (Id.). Phages have evolved diverse mechanisms to overcome restriction systems, such as the temperate coliphage P1 encoding virion-associated proteins that protect its DNA from host type I R-M systems (Id.). The P1 Dar (defense against restriction) system is comprised of at least six virion-associated proteins: DarB, Ulx, Hdf, DarA, DdrA, and DdrB, wherein DarB protects P1 DNA from EcoB and EcoK restriction in cis by an unknown mechanism and is incorporated into the virions only in the presence of Hdf, DarA, and DdrA (Id.). Kumar also discloses truncated and tagged variants of DarB (pp. 99-100, & 121-128).
It would have been obvious to one of ordinary skill in the art to modify the bacteriophage BcepIL02 or Bcep22 genes gp75 or gp70 to swap out DarB in place of the genes’ DarB-like domains, to generate a fused gene where DarB is fused to the remainder of the gp75 or gp70. As demonstrated by Iyer, DarB is a polyvalent protein that possesses multiple domains with discreet functions. Kumar shows that genetic modification of DarB is possible and known in the art, since Kumar discloses a number of DarB truncations and a HIS-tagged DarB. Because DarB relies on other genes to properly function, it also would have obvious to one of ordinary skill in the art to further include the other Dar genes, such as DarA, Ulx, Hdf, DdrA, and DdrB into the modified bacteriophage.
Gill discloses that DarB, gp75, and gp70 (DarB-like proteins) perform the same function, are virion associated, and contain a methyltransferase domain followed by a DExH helicase domain. However, the Dar-B like proteins gp75 and gp70 further encode a lytic transglycosylase domain. As disclosed by Fokine, lytic transglycosylases are enzymes that act on peptidoglycan to facilitate release of phage progeny.
“It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art.” In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980) (citations omitted).
Claim 7 recites “lysozyme fused to DarB”. In interpreting the limitation “lysozyme”, the examiner relies on the specification, which indicates that “due to the lysozyme fused-type DarB protein in which lysozyme is fused, the bacteriophage of the invention can avoid the restriction mechanism of the host bacterium, replicate its own genetic information efficiently in the host, lyse the host bacterium efficiently and grow efficiently. Examples of such a fused gene include gp70 (YP_002922742.1) of Burkholderia virus BcepIL02, gp75 (44303.1) of Burkholderia virus Bcep22” (spec., p. 3). Although Gill refers to these enzymes’ domains as “lytic transglycosylase” rather than “lysozyme” (Gill, p. 5308, col. 2, para. 3), the examiner is interpreting “lytic transglycosylase” to read on the claim term “lysozyme”, due to the instant specification.
One of ordinary skill in the art would have been motivated to provide the resulting bacteriophage with both the function of DarB and also lytic transglycosylase gp75 or gp70 activity.
There would be a reasonable expectation of success because DarB is known to be amenable to engineering, and DarB and DarB-like proteins have overlapping purposes, to protect phage DNA against restriction systems.
Therefore, claims 7, 10, 11, 13, and 17 were prima facie obvious before the priority date of the instant invention.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Gill, Fokine, Iyer, and Kumar (supra) as applied to claims 7, 10, 11, 13, and 17 above, and further in view of Summer, et al. (J Mol Biol. 2007 Nov 9;373(5):1098-112. doi: 10.1016/j.jmb.2007.08.045. Epub 2007 Aug 24. PMID: 17900620; hereinafter referred to as “Summer”).
In a more specific embodiment, the bacteriophage further comprises genes encoding three lytic enzymes including endolysin, the lysozyme fused to DarB, and Rz/Rzl (claim 8).
The Prior Art
The teachings of Gill, Fokine, Iyer, and Kumar are described above. However, they do not teach the bacteriophage further comprises genes encoding three lytic enzymes including endolysin, the lysozyme fused to DarB, and Rz/Rz1.
Summer discloses that bacteriophage λ Rz and Rz1 genes are lysis genes and remarkable because Rz1, encoding an outer membrane lipoprotein, is completely embedded in the +1 register within Rz, which itself encodes an integral inner membrane protein (Abstract). Summer further discloses that Rz/Rz1 equivalents are nearly ubiquitous among phages of Gram-negative hosts, with 120 of 137 phages possessing genes that fit the search criteria (Id.). In fact, Rz/Rz1 genes, or their spanin equivalent, are present in the overwhelming majority of tailed phages and tectiviruses of Gram-negative hosts (p. 1107, col. 2, para. 2). Summer further discloses that Rz/Rz1 proteins may have a more significant role than previously contemplated for Rz/Rz1 proteins in the general phenomenon of lysis timing (p. 1109, col. 1, para. 1). Summer also discloses construction of λ Rz/Rz1 genes into an expression plasmid (p. 1109, col. 2, para. 3).
It would have been obvious to one of ordinary skill in the art to modify the bacteriophage comprising a gene encoding lysozyme fused to DarB to further comprise genes encoding including endolysin, and Rz/Rz1. Gill discloses that Bcep22 and BcepIL02 encode endolysin and Rz/Rz1-like encoding genes. As described above, it would have been obvious to construct a bacteriophage of Bcep22 or BcepIL02 backbone with a lysozyme fused to DarB. Summer discloses that Rz/Rz1 are lysis proteins that may have a significant role in lysis timing. It also would have been obvious to one of ordinary skill in the art to replace the Rz/Rz1-like proteins encoding gene with Rz/Rz1, since they are similar genes which presumably serve the same purpose.
“It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art.” In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980) (citations omitted).
One of ordinary skill in the art would have been motivated to provide the bacteriophage with multiple genes for lysis of host bacteria.
There would be a reasonable expectation of success because natural phages contain closely related lysis cassettes with the canonical gene order of holin-endolysin-Rz/Rz1, and genetic manipulation of DarB with fusions and truncations is known in the art.
Therefore, claim 8 was prima facie obvious before the priority date of the instant invention.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Gill, Fokine, Iyer, Kumar, and Summer (supra), as applied to claim 8 above, and further in view of Sobrero, et al. (Front Mol Biosci. 2020 Jul 10;7:127. doi: 10.3389/fmolb.2020.00127. PMID: 32754614; hereinafter referred to as “Sobrero”).
Alternatively, the bacteriophage further comprises genes encoding Rz/Rzl, CsrA, and two tail fiber proteins (claim 9).
The Prior Art
The teachings of Gill, Fokine, Iyer, Kumar, and Summer are described above. However, they do not teach the bacteriophage further comprises genes encoding Rz/Rzl, CsrA, and two tail fiber proteins.
Sobrero discloses that members of the CsrA family are small dimeric proteins that act as global regulators of gene expression because they recognize characteristic sequence/structural motifs (short hairpins with GGA triplets in the loop) present in hundreds of cellular mRNAs (p. 1; p. 15, col. 2, last para.; Abstract). Sobrero further discloses that a number of phages encode CsrA homologs, including phage BcepIL02 (Table 3; p. 15, col. 2). Sobrero also suggests that CsrA proteins are instrumental for the fitness of mobile genetic elements (p. 17, col. 1, para. 1).
It would have been obvious to one of ordinary skill in the art to modify a Bcep22 or BcepIL02 bacteriophage comprising a gene encoding lysozyme fused to DarB to swap in CsrA and Rz/Rz1 encoding genes. Gill discloses that phage Bcep22 and BcepIL02 encode CsrA-like regulators, Rz/Rz1-like proteins, and multiple putative tail fiber protein orthologs.
“It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art.” In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980) (citations omitted).
One of ordinary skill in the art would have been motivated to introduce Rz/Rz1 and CsrA genes that are homologous to its cognate genes, which presumably serve the same purpose.
There would be a reasonable expectation of success because phages BcepIL02 and Bcep22 encode Rz/Rz1 and CsrA homologs and DarB is known to be amenable to genetic manipulation such as truncation and tagging.
Therefore, claim 9 was prima facie obvious before the priority date of the instant invention.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Gill, Fokine, Iyer, and Kumar (supra) as applied to claims 7, 10, 11, 13, and 17 above, and further in view of Wang, et al. (Arch Virol. 2019 Sep;164(9):2339-2343. Epub 2019 Jun 18. PMID: 31214785; hereinafter referred to as “Wang”).
Alternatively, the bacteriophage infects Ralstonia solanacearum (claim 15).
The Prior Art
The teachings of Gill, Iyer, Kumar, and Fokine are described above. However, they do not teach a bacteriophage that infects Ralstonia solanacearum.
Wang discloses characterization and analysis of phage GP4, a lytic Bcep22-like podovirus, which lysed 16 strains of R. solanacearum (Abstract). Wang further discloses that R. solanacearum causes severe bacterial wilt in more than 400 plant species, including important crops, and in some cases may reduce crop yields by 50% (p. 2339, para. 1). Wang also discloses that phage therapy has attracted attention as a potentially viable strategy for controlling bacterial wilt, and can reduce the use of chemical agents against pathogens, thus minimizing environmental pollution and chemical residue on crops (p. 2339, col. 2). Phage GP4 has a linear, double-stranded DNA genome consisting of 61,129 bp, with a GC content of 64%, an icosahedral head with a diameter of ~60 nm, and is most closely related to phages Bcep22 and BcepIL02, podoviruses (pp. 2340-41, bridging para.; Fig. 1). Phage GP4 ORF68 is a virion-associated protein that is a DarB-like antirestriction factor (p. 2341, para. 2).
It would have been obvious to one of ordinary skill in the art to modify the bacteriophage comprising a gene encoding lysozyme fused to DarB to infect Ralstonia solanacearum. Wang teaches that GP4, a phage related to BcepIL02 and Bcep22, infects Ralstonia solanacearum. Wang further discloses that GP4 encodes ORF68, a DarB-like antirestriction protein. Specifically, it would have been obvious to one of ordinary skill in the art to introduce a gene encoding lysozyme fused with DarB into the genome of phage GP4, in order to protect the GP4 genome from restriction and provide a peptidoglycan cleaving factor.
“It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art.” In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980) (citations omitted).
One of ordinary skill in the art would have been motivated to introduce a gene encoding lysozyme fused with DarB into the genome of phage GP4, in order to protect the GP4 genome from restriction and provide a peptidoglycan cleaving factor.
There would be a reasonable expectation of success because phage GP4 infects a number of Ralstonia solanacearum strains.
Therefore, claim 15 was prima facie obvious before the priority date of the instant invention.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Gill, Fokine, Iyer, Kumar, and Wang (supra) as applied to claim 15 above, and further in view of JP 2007252351 A (published 10/4/2007, machine translation due to original document being in Japanese language; hereinafter referred to as “‘351”).
In one embodiment of the claimed invention, the Ralstonia solanacearum is strain MAFF211270, strain MAFF730139, strain MAFF211272, or strain MAFF301558 (claim 16).
The Prior Art
The teachings of Gill, Fokine, Iyer, Kumar, and Wang are described above. However, they do not teach a Ralstonia solanacearum strain MAFF211270, strain MAFF730139, strain MAFF211272, or strain MAFF301558.
‘351 teaches a means for effectively controlling Ralstonia solanacearum, where a bacteriophage showing bacteriolytic action to strains MAFF211270, MAFF730139, MAFF211272, and MAFF301558 is utilized (Abstract). ‘351 further discloses a bacteriophage, RSA1, which exhibits a broad lytic spectrum against bacterial wilt from Ralstonia solanacearum (pp. 1-2).
It would have been obvious to one of ordinary skill in the art to modify the teachings of Gill, Fokine, Iyer, Kumar, and Wang to use a bacteriophage comprising a gene encoding lysozyme fused to DarB to infect Ralstonia solanacearum strains MAFF211270, MAFF730139, MAFF211272, or MAFF301558. It would have been obvious to generate an RSA1 phage bearing a gene encoding lysozyme fused to DarB (such as BcepIL02 or Bcep22 DarB-like lytic transglycosylase domain fused to DarB.
One of ordinary skill in the art would have been motivated to provide antirestriction and lytic transglycosylase activity to the RSA1 phage.
There would be a reasonable expectation of success because RSA1 shows bacteriolytic activity toward strains MAFF211270, MAFF730139, MAFF211272, and MAFF301558 of Ralstonia solanacearum, and DarB and DarB-like proteins exhibit antirestriction activity.
Therefore, claim 16 was prima facie obvious before the priority date of the instant invention.
Claims 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over Gill, Fokine, Iyer, Kumar, Wang, and ‘351 (supra) as applied to claim 16 above, and further in view of Murthy, et al. (WO 2006047870 A1, published 5/11/2006, priority date 11/2/2004; hereinafter referred to as “Murthy”).
Another embodiment of the claimed invention encompasses a composition comprising the bacteriophage comprising a gene encoding lysozyme fused to DarB and a pharmacologically and botanically acceptable protein stabilizer (claim 18), which prevents wilt by Ralstonia solanacearum when applied to a plant (claim 19). In a specific embodiment, the bacteriophage is present at a concentration ranging from 103 to 1012 pfu/mL (claim 20).
The Prior Art
The teachings of Gill, Fokine, Iyer, Kumar, Wang, and ‘351 are described above. ‘351 also discloses the phage concentration in a soil solution is preferably 1x105 to 1x1011 pfu/ml for use as a bacterial wilt control agent (p. 3). ‘351’s compositions and methods are a means for effectively controlling Ralstonia solanacearum, and show bacteriolytic action to Ralstonia strains (Abstract). However, they do not teach a composition comprising a bacteriophage comprising a gene encoding lysozyme fused to DarB and pharmacologically and botanically acceptable protein stabilizer.
Murthy discloses stabilized bacteriophage compositions for use as antibacterials that may be used for treatment of plants (para. [0017]; Abstract), and that the antibacterial composition can be adsorbed onto a matrix to produce a stabilized composition, wherein the matrix may be skim milk powder, plant peptone, or a number of other protein powders (Claims 1-2). Murthy discloses that stabilized bacteriophage formulations may be used to control the multiplication of various strains of bacteria, but to be commercially viable, the bacteriophages themselves must show a certain degree of stability to allow for storage (para. [0002]).
It would have been obvious to one of ordinary skill in the art to modify the teachings of Gill, Fokine, Iyer, Kumar, Wang, and ‘351 to use a pharmacologically and botanically acceptable protein stabilizer such as those taught by Murthy, skim milk powder, plant peptone, or a number of other protein powders. Additionally, Wang and ‘351 disclose use of phages to treat wilt caused by Ralstonia solanacearum, and ‘351 further discloses a phage concentration in a soil solution is preferably 1x105 to 1x1011 pfu/ml.
One of ordinary skill in the art would have been motivated to provide stabilize the phage compositions because it would allow the bacteriophages to be stored and remain commercially viable.
There would be a reasonable expectation of success because Murthy demonstrates the applicability of such stabilizers in phage compositions.
Therefore, claims 18-20 were prima facie obvious before the priority date of the instant invention.
Conclusion
No claim is allowed.
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/JEFFREY MARK SIFFORD/Examiner, Art Unit 1671 /Michael Allen/Supervisory Patent Examiner, Art Unit 1671