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has been timely paid, the finality of the previous Office action has been withdrawn pursuant to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 12/30/2025 has been entered.
Receipt is acknowledged of an amendment, 12/30/2025, in which claims 1 and 8 were amended and claims 13-15, 19 and 20 were previously withdrawn due to the previous restriction requirement. Claims 1-12 and 16-18 are currently under examination.
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Claims 1-12 and 16-18 are rejected under 35 U.S.C. 103 as being unpatentable over Biryukova et al (WO 2006/001514 Al), in view of Sanchez-Romero et al (Appl Environ Microbiol; 1998) and Herring (Gene 331, Pgs. 153-163; 2003) and further in view of Rodriguez-Garcia et al (Nucleic Acid Research; Vol. 33; 2005). This rejection was made in the Office action mailed 08/04/2025 and has been rewritten to address the amendment to the claims in the reply filed 12/30/2025.
Regarding Claim 1, Biryukova teaches a vector for the purpose of genome manipulation in a bacteria host cell where the vector includes the replacement of the antibiotic resistance genes in RSF1010mob- plasmid to result in RSF1010-MT, which is discussed in Example 5 (Page 12 and Page 25, Example 5). Biryukova teaches in Fig. 5, the plasmid map including the thyA gene operably linked to the promoter (Page 33, Figure 5). Biryukova also teaches that the promoter that is linked to the thyA gene has been modified to have a perfect Pribnow-box (Page 24). Biryukova teaches an origin of replication denoted as oriV, which is a unique origin of vegetative DNA replication (Page 7). Biryukova teaches in figure 5 that the oriV is in the RSF1010-MT plasmid (Page 33, figure 5). Biryukova teaches the restriction enzyme recognition sites for PdtI, EcoRI and NotI restriction endonucleases (Page 33, Figure 5). Biryukova teaches in example 4 the use of EcoRI and NotI restrictases for subcloning into corresponding sites of the RSF1010mob- plasmid (Page 25, Example 4).
Biryukova does not teach the origin of replication which is not capable of inducing replication of the plasmid within the bacteria as well as the meganuclease and gene encoding the meganuclease is on a single plasmid together.
Sanchez-Romero teaches that the basic replicon of this plasmid contains the origin of replication (oriV) that is only able to replicate in Pseudomonas bacteria unless altered in some way (4040; column 2, paragraph 2). Sanchez-Romero teaches that even low levels of expression result in high resistance, meaning that the vector would not need to replicated indefinitely to achieve the purpose of resistance (Page 4040; Column 2).
Sanchez-Romero does not teach the meganuclease and gene encoding the meganuclease is on a single plasmid together.
Herring teaches a two-plasmid system where one plasmid contains the I-Sce I gene and the other contained the I-Sce I recognition site (Page 158, Figure 2). Herring teaches the introduction of the two-plasmid system together to produce co-transformants and where the cells will contain both plasmids (Page 156; Column 1). It would have been obvious to combine the I-Sce I gene and restriction sites on the same plasmid in order to only have to do one transformation instead of two. Herring teaches recombination is accomplished by the Red recombination system encoded by the bacteriophage lambda genes, such as gam, bet and exo, which operates on linear DNA (Page 153, Column 2 and Page 154, Column 1).
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 teachings of Biryukova to include the regulatory sequence operatively linked to the gene in order to regulate the expression of the gene taught by Sanchez-Romero because Biryukova teaches it is within the ordinary skill in the art to use non-antibiotic selection markers within a vector cassette for the purpose of gene manipulation and Sanchez-Romero teaches that the origin of replication that would not be able to replicate outside of the Pseudomonas bacteria as well as a regulatory sequence operably linked to a non-antibiotic selection marker. The specification of the current invention points to the origin of replication and its ability of being not capable of replication is based solely on the type of bacterium selected for genetic manipulation (Page 8; Lines 23-26).
It would have also been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Biryukova and Sanchez-Romero to include the restriction endonuclease from the LAGLIDADG family, I-SceI, taught by Herring because Biryukova and Sanchez-Romero teaches it is within the ordinary skill in the art to use a restriction endonuclease for the purpose of cleaving DNA at the recognition site and Herring teaches I-SceI is a strong endonuclease capable of completing the cut at the recognition site. It would have been obvious to combine the components onto a single vector to simplify administration, reduce steps, or improve efficiency, yielding predictable results (See MPEP § 2144.04).
One would have been motivated to make such a modification in order to receive the expected benefit of controlled expression of the gene of interest without excessive replication as taught by Sanchez-Romero as well as the expected benefit of the known properties of the I-SceI restriction endonuclease and its function to properly identify and cut and the recognition site as taught by Herring.
Biryukova, Sanchez-Romero and Herring do not teach a second regulatory sequence being a promoter inducible by tetracycline or a variant of tetracycline such as anhydrotetracycline, minocycline, metacycline, sanocycline, demeclocycline, chloro-tetracycline, oxytetracycline, doxycycline, or tigecycline.
Rodriguez-Garcia teaches the use of a promoter induced by anhydrotetracycline for the purpose of increased expression when activated (abstract). Specifically, the tcp830 promoter used with resistance marker genes (Page 5; Column 1).
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 teachings of Biryukova and Sanchez-Romero to include the promoter inducible by a variant of tetracycline taught by Rodriguez-Garcia because Biryukova and Sanchez-Romero teaches it is within the ordinary skill in the art to use an expression vector for the purpose of gene modification containing a promoter for regulation of gene expression, Herring teaches I-SceI is a strong endonuclease capable of completing the cut at the recognition site and Rodriguez-Garcia teaches that a promoter inducible by anhydrotetracycline is a strong promoter when induced to express higher levels of resistance from the target resistance gene.
One would have been motivated to make such a modification in order to receive the expected benefit of a strong promoter for increased expression levels of resistance from the resistance gene as taught by Rodriguez-Garcia.
Regarding Claims 2 and 3, Herring teaches the Rec mediated single homologous crossover results in cointegration of the whole circle into the genome at the target site with the plasmid vector between a wild type and mutant copy of the target sequence (Page 153, Column 2). Herring teaches the plasmid is capable of facilitating double recombination at a position on the chromosome homologous to the donor sequence (Page 157; Column 2). Herring teaches that the system generates mutants at a relatively high frequencies without direct selection (Page 159; Column 1 bridging column 2). Herring teaches the use of two I-Sce I sites where both ends of the fragment match the chromosomal gene (Page 161, Column 2).
Regarding Claims 4-6, Sanchez teaches the use of tellurite resistance gene as the selective marker which belongs to the heavy metal resistance gene group (Page 4040; Column 1). Sanchez teaches the use of resistance to tellurite salts as a selective marker as a resistance gene for the purpose of environmental release. (Page 4040; column 2). Sanchez-Romero teaches that the resistance to potassium tellurite as a selection phenotype is an attractive marker, because it is toxic for most microorganisms, especially gram-negative bacteria, and spontaneous tolerance is very infrequent (Page 4042, column).
Regarding Claims 7, 17 and 18, Biryukova teaches the use of the tdk gene as the non-antibiotic selection marker which is denoted as SEQ ID NO: 46 to be 618 bp long which corresponds to SEQ ID NO: 5 in the current invention (Page 25).
Regarding claims 8 and 9, Herring teaches a plasmid that is linearized by in vivo expression of the meganuclease I-Sce I, providing a DNA substrate for lambda Red mediated recombination (Abstract). Herring teaches a two-plasmid system where one plasmid contains the I-Sce I gene and the other contained the I-Sce I recognition site (Page 158, Figure 2). Herring teaches the introduction of the two-plasmid system together to produce co-transformants and where the cells will contain both plasmids (Page 156; Column 1). It would have been obvious to combine the I-Sce I gene and restriction sites on the same plasmid in order to only have to do one transformation instead of two. It is known in the art that I-SceI endonucleases are a part of the LAGLIDADG family of endonucleases.
Regarding Claims 10 and 11, Biryukova teaches the use of the host bacteria cell to be from the Enterobacteriaceae family (Page 15). More specifically, the host bacteria used was Escherichia coli (page 21; example 3).
Regarding Claim 12, Biryukova, Sanchez-Romero and Herring do not teach a second regulatory sequence being a promoter inducible by tetracycline or a variant of tetracycline such as anhydrotetracycline, minocycline, metacycline, sanocycline, demeclocycline, chloro-tetracycline, oxytetracycline, doxycycline, or tigecycline.
Rodriguez-Garcia teaches the use of a promoter induced by anhydrotetracycline for the purpose of increased expression when activated (abstract). Specifically, the tcp830 promoter used with resistance marker genes (Page 5; Column 1).
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 teachings of Biryukova and Sanchez-Romero to include the promoter inducible by a variant of tetracycline taught by Rodriguez-Garcia because Biryukova and Sanchez-Romero teaches it is within the ordinary skill in the art to use an expression vector for the purpose of gene modification containing a promoter for regulation of gene expression and Rodriguez-Garcia teaches that a promoter inducible by anhydrotetracycline is a strong promoter when induced to express higher levels of resistance from the target resistance gene.
One would have been motivated to make such a modification in order to receive the expected benefit of a strong promoter for increased expression levels of resistance from the resistance gene as taught by Rodriguez-Garcia.
Regarding Claim 16, Sanchez-Romero teaches that the resistance to potassium tellurite as a selection phenotype is an attractive marker, because it is toxic for most microorganisms, especially gram-negative bacteria, and spontaneous tolerance is very infrequent (Page 4042, column).
Response to Arguments - Claim Rejections - 35 USC § 103
The rejection of claims 1-11 and 16-18 under 35 U.S.C. 103 as being unpatentable over Biryukova et al (WO 2006/001514 A1), in view of Sanchez-Romero et al (Appl Environ Microbiol; 1998) and Herring (Gene 331, Pgs. 153-163; 2003) and the rejection of claim 12 under 35 U.S.C. 103 as being unpatentable over Biryukova, Sanchez-Romero and Herring as applied to claim 1-11 and 16-18 above, and further in view of Rodriguez-Garcia et al (Nucleic Acid Research; Vol. 33; 2005), has been maintained and rewritten as stated above. The previous rejection has been re-written to include the rejection of claim 12 under 35 U.S.C. 103 as being unpatentable over Biryukova et al (WO 2006/001514 A1), in view of Sanchez-Romero et al (Appl Environ Microbiol; 1998) and Herring (Gene 331, Pgs. 153-163; 2003) and further in view of Rodriguez-Garcia et al (Nucleic Acid Research; Vol. 33; 2005) as Applicant amended claim 1 to include the dependent claim limitation of claim 12.
Applicant’s arguments filed 12/30/2025 have been fully considered but they are not found persuasive.
Applicant argues the combination of the cited references fail to teach or suggest this novel and non-obvious configuration of an integrated, single vector counter selection system because the cited references provide no motivation to combine the features. Specifically, Applicant argues that the claimed vector is structurally distinct from the system of Herring in that Herring requires two vectors while the currently claimed invention only requires one and no teaching or suggestion of Herring provides a motivation to combine the components onto a single plasmid.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that 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, the cited references are used in combination to teach the claimed invention. Such that, Biryukova teaches it is within the ordinary skill in the art to use non-antibiotic selection markers within a vector cassette for the purpose of gene manipulation, Sanchez-Romero teaches that the origin of replication that would not be able to replicate outside of the Pseudomonas bacteria as well as a regulatory sequence operably linked to a non-antibiotic selection marker and Herring teaches I-SceI is a strong endonuclease capable of completing the cut at the recognition site. It would have been obvious to combine the components onto a single vector to simplify administration, reduce steps, or improve efficiency, yielding predictable results (See MPEP § 2144.04). One would have been motivated to make such a modification in order to receive the expected benefit of controlled expression of the gene of interest without excessive replication as taught by Sanchez-Romero as well as the expected benefit of the known properties of the I-SceI restriction endonuclease and its function to properly identify and cut and the recognition site as taught by Herring.
Applicant continues to argue that the instant claims require an inducible promoter and Rodriguez-Garcia does not meet the limitation of the claims due to the reference using the inducible promoter for the control of a resistance gene in a system designed to promote cell survival and confer a benefit. Applicant continues to argue that the instant claimed invention uses the inducible promoter that controls expression of the meganuclease gene which functions as a counter selection “kill switch”. Applicant that this argument extends to claim 12 which requires the use of a promoter induced by anhydrotetracycline.
However, the instant claims do not recite the specific limitation of the promoter induced by anhydrotetracycline for the intended use of activation of a “kill switch”. The promoter induced by anhydrotetracycline would be an activation of the meganuclease gene which would confer the intended use as a “kill switch”. For these reasons, the meganuclease gene being activated by any regulatory element would have the intended use as a “kill switch” and not the inducible promoter itself. Rodriguez-Garcia teaches the use of a promoter induced by anhydrotetracycline for the purpose of increased expression when activated (abstract). Specifically, the tcp830 promoter is an inducible promoter induced by the presence of anhydrotetracycline (Page 5; Column 1). The “increased expression when activated” would be of the meganuclease gene which would have the function of a “kill switch”. However, this intended use recited (“kill switch”) is not a current limitation of the instant claims.
Regarding claims 6 and 16, Applicant argues that Biryukova’s thyA selection system is not a simple non-antibiotic marker due to it only functioning in bacterial strains that have been pre-engineered to lack both the thyA and tdk genes whereas in contrast, the tellurite/tpm system instantly claimed does not require such preliminary specific strain engineering, making it a simpler and more broadly applicable system.
However, these are features not specifically claimed in the instant claims. The instant claims do not limit that the premodification can not accomplished nor does Applicant provide evidence of unexpected or improved results of how the system is a simpler and more broadly applicable system due to not having the preliminary specific strain engineering making it more advantageous over the cited prior art.
Regarding claims 8 and 9, Applicant argue the same opinion that the combination of the cited references fails to teach or suggest this novel and non-obvious configuration of an integrated, single vector counter selection system because the cited references provide no motivation to combine the features. Specifically, Applicant argues that the claimed vector is structurally distinct from the system of Herring in that Herring requires two vectors while the currently claimed invention only requires one and no teaching or suggestion of Herring provides a motivation to combine the components onto a single plasmid.
However, and as stated above, in response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that 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, the cited references are used in combination to teach the claimed invention. Such that, Biryukova teaches it is within the ordinary skill in the art to use non-antibiotic selection markers within a vector cassette for the purpose of gene manipulation, Sanchez-Romero teaches that the origin of replication that would not be able to replicate outside of the Pseudomonas bacteria as well as a regulatory sequence operably linked to a non-antibiotic selection marker and Herring teaches I-SceI is a strong endonuclease capable of completing the cut at the recognition site. It would have been obvious to combine the components onto a single vector to simplify administration, reduce steps, or improve efficiency, yielding predictable results (See MPEP § 2144.04). One would have been motivated to make such a modification in order to receive the expected benefit of controlled expression of the gene of interest without excessive replication as taught by Sanchez-Romero as well as the expected benefit of the known properties of the I-SceI restriction endonuclease and its function to properly identify and cut and the recognition site as taught by Herring.
Conclusion
No claims are allowed.
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/ALEXANDRA ROSE LIPPOLIS/ Examiner, Art Unit 1637
/CELINE X QIAN/ Primary Examiner, Art Unit 1637