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 .
Application Status
This action is written in response to applicant’s correspondence received 7 April 2026. Claims 1 and 109-143 are currently pending. Claims 116-117 and 122-143 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1, 109-115, and 118-121 are examined herein. The restriction requirement mailed 25 October 2024 is still deemed proper. Applicant's elected Group I, claims 1 and 109-121, alongside the species of L. gasseri, the selectable maker gene sequence, counterselection, Lactobacilli, and a protein involved in bacterial adherence without traverse in the reply filed 6 December 2024.
Any rejection or objection not reiterated herein has been overcome by amendment.
Applicant' s 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 § 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.
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(s) 1, 109, 111, 119, and 121 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lambert (Applied and environmental microbiology 73.4 (2007): 1126-1135) in view of Campo (Applied and environmental microbiology 68.5 (2002): 2359-2367). This rejection is maintained.
Regarding claim 1, Lambert is drawn towards a study concerned with a Cre-lox based system for multiple gene deletions and selectable marker removal in Lactobacillus plantarum (Abstract). Lambert teaches the use of a circular vector that comprises (a) an erythromycin resistance gene under control of a P32 promoter (i.e., a selectable marker) and (b) rare-cutting blunt-end restriction sites SwaI, PmeI, SrfI, and Ecl136II that allow for direct cloning of blunt-end PCR products (i.e., a multiple cloning site) that are flanked by lox71 and lox66 sites (i.e., a first region flanked on either side by a target site for a site-specific recombinase) (pg. 1130; see FIG. 2). Lambert teaches that a P32-cat gene was present on the vector and not within the first region (pg. 1130; see FIG. 2). Lambert teaches that the P32-cat gene was able to replace target genes of interest via homologous recombination and can later be excised from the genome when desired (pg. 1131; see FIG. 3). Lambert teaches the use of a second vector, pNZ5348, that expresses a Cre recombinase (i.e., a site-specific recombinase) and comprises a replicon sequence (pg. 1130). Lambert teaches that more advanced alternative possibilities could also be employed, including expression of Cre from a plasmid in which Cre or the origin of replication is flanked by lox sites to ensure cessation of Cre activity before construction of additional mutations in the same genetic background (pg. 1134).
Lambert does not teach or suggest that the vector further comprises a sequence encoding a site-specific recombinase operably linked to an inducible promoter, or a replicon sequence, wherein the inducible promoter and replicon sequence are not within first said region (Claim 1).
However, one of ordinary skill in the art would have considered the teachings of Campo as both references are common fields of endeavor pertaining to the use of Cre recombinases.
Campo is drawn towards a study concerned with a Cre-loxP recombination system to generate chromosomal rearrangements in L. lactis (Abstract). Campo teaches the use of a circular vector, termed pGH-Cre, that comprises a Cre recombinase under the control of a nisin-inducible promoter, PnisA, and a thermosensitive repA replicon (i.e., a replicon sequence) (pg. 2360-2362; see Table 1 and Fig. 2B). Campo teaches that, in permissive conditions for pGh-Cre replication and Cre expression, the Cre recombinase catalyzes recombination between two loxP sites (pg. 2363).
Therefore, 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 vector sequence not present within the first region of Lambert for a vector sequence encoding a Cre recombinase under control of an inducible promoter and a replicon sequence, as described by Campo. A person of ordinary skill in the art would have been motivated to do so in order to express the Cre recombinase under certain conditions and from a target genomic region of interest within a cell when desired. A person of ordinary skill in the art would have had a reasonable expectation of success because Lambert teaches that the vector sequence not present within the first region may comprise a P32-cat gene that can be integrated into, expressed from, and excised from, a target genomic region of interest when desired and indicates that the expressing of Cre from a construct in which Cre or the origin of replication is flanked by lox sites can ensure cessation of Cre activity before construction of additional mutations in the same genetic background while Campo teaches the use of a vector sequence that encodes a Cre protein and further comprises a replicon sequence.
Regarding claim 109, Lambert teaches the use of an erythromycin resistance gene (pg. 1130; see FIG. 2).
Regarding claim 111, Campo teaches that the recombinase is a Cre recombinase (pg. 2360-2362; see Table 1 and Fig. 2B).
Regarding claim 119, Campo teaches that the inducible promoter is a nisin inducible promoter (pg. 2360-2362; see Table 1 and Fig. 2B)
Regarding claim 121, Lambert teaches that the presence of PmeI, SwaI, SrfI, and Ecl136II rare-cutting blunt-end restriction sites in the vector (i.e., the multiple cloning site) allows for cloning of PCR-amplified homologous DNA fragments (i.e., gene targeting sequences) that are upstream and upstream and downstream of a mutagenesis locus, which is necessary for targeting of the vector to the genomic locus of interest (pg. 1129).
Claim 110 is rejected under 35 U.S.C. 103 as being unpatentable over Lambert (Applied and environmental microbiology 73.4 (2007): 1126-1135) in view of Campo (Applied and environmental microbiology 68.5 (2002): 2359-2367) as applied to claims 1, 109, 111, 119, and 121 above, and further in view of Keravala (Molecular Genetics and Genomics 276 (2006): 135-146). This rejection is maintained.
Regarding claim 110, Lambert in view of Campo renders obvious claims 1, 109, 111, 119, and 121 as described above.
Lambert in view of Campo does not teach or suggest the use of a site-specific serine recombinase (Claim 110).
However, one of ordinary skill in the art would have considered the teachings of Keravala as both references are analogous prior art pertaining to the design of vectors comprising site-specific recombinases.
Keravala is drawn to a study concerned with the ability of five serine phage integrases, from phages A118, U153, Bxb1, φFC1, and φRV1, to mediate recombination in mammalian cells (Abstract). Keravala teaches the use of a plasmid comprising a lacZ gene flanked by attP and attB target sites for a serine site-specific recombinase (pg. 137; see Fig. 1). Keravala teaches that φFC1 (i.e., a site-specific serine recombinase) can interact with the attP and attB target sites in order to excise the lacZ gene from the plasmid (pg. 138).
Therefore, 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 Cre site-specific recombinase and lox sites of Lambert in view of Campo for a φFC1 serine site-specific recombinase and attP and attB target sites, as described by Keravala. A person of ordinary skill in the art would have had a reasonable expectation of success because both Keravala and Lambert in view of Campo teach the successful use of a site-specific recombinase that can excise a motif of interest from a plasmid via the use of two target sites.
Claim(s) 112-115 and 118 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lambert (Applied and environmental microbiology 73.4 (2007): 1126-1135) in view of Campo (Applied and environmental microbiology 68.5 (2002): 2359-2367) as applied to claims 1, 109, 111, 119, and 121 above, and further in view of Russell (Applied and Environmental Microbiology 67.9 (2001): 4361-4364). This rejection is maintained.
Regarding claims 112-115 and 118, Lambert in view of Campo renders obvious claims 1, 109, 111, 119, and 121 as described above.
Lambert in view of Campo does not specifically teach or suggest that the replicon sequence is a prokaryotic replicon sequence (Claim 112). Lambert in view of Campo does not specifically teach or suggest that the replicon sequence encodes an origin of replication (ori) and replication initiator protein (Rep protein) (Claim 113). Lambert in view of Campo does not specifically teach or suggest that the replicon sequence is a replicon sequence permissive for replication of said vector in a prokaryote host cell (Claim 114), selected from Lactobacilli (Claim 115), or Lactobacillus gasseri (Claim 118).
However, one of ordinary skill in the art would have considered the teachings of Russell as both references are common fields of endeavor pertaining to the use of RepA replicon sequences.
Russell is drawn to a study concerned with an efficient method for the generation of site- specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri (Abstract). Russell teaches the use of a plasmid, pTRK669, that comprises an origin of replication and repAC genes (i.e., a replicon sequence) (pg. 4361). Russell teaches that replication of the vector was successfully observed in L. gasseri at 37°C (i.e., Russell is interpreted as teaching the use of a replicon sequence that is permissive for replication in a Lactobacillus gasseri host cell) (pg. 4361).
Therefore, 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 RepA replicon sequence rendered obvious by Lambert in view of Campo for a repAC replicon sequence, as described by Russell. A person of ordinary skill in the art would have had a reasonable expectation of success because Lambert in view of Campo renders obvious the use of a repA replicon sequence that can be utilized to modify bacterial chromosomes and Russell teaches the use of a repAC replicon sequence that can be utilized in bacterial cells.
Claim 120 is rejected under 35 U.S.C. 103 as being unpatentable over Lambert (Applied and environmental microbiology 73.4 (2007): 1126-1135) in view of Campo (Applied and environmental microbiology 68.5 (2002): 2359-2367) as applied to claims 1, 109, 111, 119, and 121 above, and further in view of Cheo (PG Pub No. US 2002/0007051 A1). This rejection is maintained.
Regarding claim 120, Lambert in view of Campo renders obvious claims 1, 109, 111, 119, and 121 as described above.
Lambert in view of Campo does not teach or suggest that the two target sites for said site-specific recombinase are orientated such that the product of site-specific recombination between two target sites for said site-specific recombinase is a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d) (Claim 120).
However, one of ordinary skill in the art would have considered the teachings of Cheo as both references are analogous prior art pertaining to the use of vectors comprising lox sites.
Cheo is drawn to an invention concerned with compositions and methods for recombinational cloning (Abstract). Cheo teaches the use of a single large plasmid comprising motifs that are flanked by loxP sites ([0237]; see FIG. 27A). Cheo teaches that addition of a Cre protein resolves the single large plasmid into two smaller ones (i.e., a first and second plasmid) ([0237]; see FIG. 27A). Cheo teaches that the first plasmid comprises motifs that are located outside of the loxP sites while the second plasmid comprises motifs that are located within the loxP sites ([0237]; see FIG. 27A). Cheo teaches that utilizing Cre and loxP sites in order to split a single plasmid into two different plasmids allows for the loss of undesired motifs present within the loxP sites while simultaneously maintaining desired motifs located outside of the loxP sites ([0237]).
Therefore, 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 vector of Lambert in view of Campo such that the two target sites for said site-specific recombinase are orientated such that the product of site-specific recombination between two target sites for said site-specific recombinase is a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d), as described by Cheo. A person of ordinary skill in the art would have been motivated to do so in order to remove (a) and (b) located within the loxP sites from the plasmid comprising (a)-(e) in order to render the plasmids unable to operate. A person of ordinary skill in the art would have had a reasonable expectation of success because both Cheo and Lambert in view of Campo teach the use of vectors comprising loxP sites that can have motifs present within the loxP sites excised from the vector.
Response to Arguments
Applicant's arguments filed 7 April 2026 have been fully considered but they are not persuasive.
Applicant alleges that the architectural groupings of the circular DNA vector of Claim 1 and the pNZ5319 vector of Lambert (see Figure 2B) are different as laid out in the Table present in the response filed (Remarks; pg. 10-11).
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Additionally, Figure 2B of Lambert is reproduced below.
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Figure 1. pNZ5319 of Lambert (see Figure 2B)
Regarding the “Flanked Region”, and the differences asserted by Applicant in the first row of the Table present on pg. 10-11 of the response filed 7 April 2026, it is noted that because the DNA vector of Lambert is circular, the claimed first region can be either the region comprising the cat gene or the region comprising the (a) ery selectable marker and the (b) multiple cloning sites for SwaI, PmeI, Xhol, Sfrl, Ecl136ll, and Bglll. If a person of ordinary skill in the art were to interpret the first flanked region of Lambert as the region comprising the ery selectable marker and the multiple cloning sites, the POSITA would appreciate that the region reads on sections (a)-(b) of the claimed circular DNA vector.
Regarding the “Backbone Region” and “Proviso Exclusion”, and the differences asserted by Applicant in the second to third rows of the Table present on pg. 10-11 of the response filed 7 April 2026, it is noted that the claimed circular DNA vector does not define sections (c)-(d) as being part of the backbone of the vector. Parts (c)-(d) of the claimed vector are only defined relative to their exclusion from the first region (see Claim 1). Under the broadest reasonable interpretation of the claim, the region of Lambert’s vector comprising the cat gene would read on the region that is excluded from the first region comprising the ery selectable marker and the multiple cloning sites.
Additionally, as described above, Lambert expressly teaches that (c) a Cre recombinase or (d) the origin of replication (i.e., a replicon sequence) can be flanked by loxP sites to ensure cessation of Cre activity before construction of additional mutations in the same genetic background. The teachings of Campo are relied upon for evidence supporting the assertion that it was known in the art that Cre can be placed under the control of an inducible promoter and present alongside a thermosensitive repA replicon sequence in order to successfully express the recombinase. Accordingly, it is the combination of Lambert and Campo that allows a POSITA to arrive at the claimed region that is excluded from the first region because Lambert suggests flanking sequences encoding a Cre recombinase and an origin of replication with loxP sites while Campo teaches that the claimed inducible promoter can be linked to a Cre recombinase and utilized alongside a replicon sequence in order to express the recombinase.
Applicant alleges that the vector of Lambert reflets and entirely different strategy and is used for marker recycling as opposed to the claimed vector, wherein the vector is positioned to “self-destruct” because the marker and replicon of Lambert are not positioned to be split into circular intermediates (Remarks; pg. 11). Applicant alleges that, upon consulting the disclosure of Lambert, the POSITA would at most be motivated to introduce the sequence encoding the recombinase in the first region together with the selectable cat marker because of the benefit comprising the following: “the sequence encoding the site-specific recombinase would be re-introduced together with the recycled marker in the second round of targeting thereby avoiding potential leaking expression of the recombinase, which would induce site-specific recombination of the Lambert vector in its episomal state during the second round of targeting” (Remarks; pg. 12). Applicant alleges that the POSITA would not separate the sequences from each other as in the claimed invention (Remarks; pg. 12). Applicant alleges that the claimed DNA vector has a different functional intent when compared to the functional intent of the Lambert vector (Remarks; pg. 11).
It is noted that the proposed modifications to the DNA vector of Lambert would not render it unsatisfactory for its intended purpose because, as discussed above, Lambert expressly teaches that Cre can be expressed from a construct in which Cre or the origin of replication is flanked by lox sites such that Cre activity can be ceased before construction of additional mutations in the same genetic background. Additionally, as discussed above, because the region of the Lambert vector comprising the cat selectable marker reads on the claimed (c)-(d) regions of the claimed vector, and as noted by Applicant the purpose of Lambert’s vector is recombination in the episomal state, a person of ordinary skill in the art would appreciate that the benefit of the proposed modification is that the sequence encoding the site-specific recombinase would be re-introduced together with the recycled marker in the second round of targeting thereby avoiding potential leaking expression of the recombinase, which would induce site-specific recombination of the Lambert vector in its episomal state during the second round of targeting. Therefore, the purpose of Lambert would be met through the integration of the Cre and replicon sequence within the region comprising the cat selectable marker.
Applicant alleges that the vector of the claimed invention is designed with a different strategy for gene targeting (Remarks; pg. 12-13). Applicant alleges that in the instant invention the site-specific recombination event resolves the episomal vector into two products: A first circular product containing the targeting sequence (and selectable marker) that cannot replicate and a second circular product containing the replication origin and recombinase (see Figure 1A of the present application) (Remarks; pg. 12). Applicant alleges that the intended use of Lambert is vector recycling, as opposed to the instant invention’s purpose of “self-destructing” (Remarks; pg. 11 and 13).
This argument is not found persuasive because, as described above, the proposed modifications do not render the vector of Lambert unsatisfactory for its intended purpose and, as noted by Applicant, provides benefits in later rounds of targeting through the inclusion of the claimed parts (c) and (d) within the region of the vector comprising the cat selectable marker. Additionally, the claimed invention is defined as a circular DNA vector comprising a first region flanked on either side by recombinase target sites and (a) and (b) with the proviso that (c) and (d) are not within said first region (see Claim 1). Thus, the arguments that the first circular product containing the targeting sequence and selectable marker cannot replicate are moot because, as taught by MPEP 211.03, “comprising” is open ended and does not exclude additional elements. Because, as discussed above, the combination of Lambert and Campo renders obvious the first region that comprises (a) and (b), alongside a second region that comprises (c) and (d), and both of these regions are separated by flanking LoxP sites in a circular DNA vector, a POSITA would recognize that the combination of Lambert and Campo renders obvious the claimed invention.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., the first region consisting of the claimed first region) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning (Remarks; pg. 14), it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Applicant alleges that Campo does not propose recombination of a vector in episomal state nor proposes expression of the recombinase in cis (Remarks; pg. 14). Applicant alleges that Campo motivates the skilled person not to abandon the strategy of providing the recombinase in trans (Remarks; pg. 14). Applicant alleges that the person of ordinary skill in the art would not separate the sequences from each other as in the claimed invention (Remarks; pg. 14).
These arguments are not found persuasive because, as described above, Lambert expressly teaches that (c) a Cre recombinase or (d) the origin of replication (i.e., a replicon sequence) can be flanked by loxP sites to ensure cessation of Cre activity before construction of additional mutations in the same genetic background. Therefore, it is the disclosure of Lambert that provides a strategy of including the (c) and (d) motifs within flanking loxP sites. As discussed above, the resulting modification to the vector of Lambert would result in the separation of (a) and (b) from (c) and (d).
Applicant alleges that there is no guidance in any one of Lambert and Campo or Cheo to separate the replication origin from a circular product comprising the MCS and marker (Remarks; pg. 15). Applicant alleges that there is no guidance or reasonable basis in Cheo to provide a vector that may be resolved into two products, where the key product is a non-replicating intermediate (Remarks; pg. 16).
This argument is not found persuasive because it is the disclosure of Lambert that provides a strategy of including the (c) and (d) motifs within flanking loxP sites. Additionally, the claimed invention is defined as a circular DNA vector comprising a first region flanked on either side by recombinase target sites and (a) and (b) with the provision that (c) and (d) are not within said first region (see Claim 1). Thus, the arguments pertaining to the separation of the replication origin from a circular product comprising the MCS and marker are moot because, as taught by MPEP 211.03, “comprising” is open ended and does not exclude additional elements. Because, as discussed above, the combination of Lambert, Campo, and Cheo renders obvious the first region that comprises (a) and (b), alongside a second region that comprises (c) and (d), and both of these regions are separated by flanking LoxP sites in a circular DNA vector, a POSITA would recognize that the combination of Lambert, Campo, and Cheo renders obvious the claimed invention.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., the first region consisting of the claimed first region, which functionally results in the claimed vector being able to separate into non-replicating intermediates) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached M-R 8:30-4:30, every other F 8:30-4:30 (EDT/EST).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil Hammell can be reached at 571-270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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