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 .
Receipt is acknowledged of an amendment, 07/09/2026, in which claims 1 and 8 were amended and claims 13-15, 19 and 20 were previously withdrawn due to the previous restriction requirement.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.
Applicant' s arguments have been thoroughly reviewed, but are not persuasive for the
reasons that follow. Any rejection and objections not reiterated in this action have been
withdrawn.
This action is FINAL.
Claim Rejections - 35 USC § 112
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 1 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. THIS IS A NEW REJECTION NECESSITATED BY AMENDMENT.
Claim 1 is vague and indefinite in that the metes and bounds of the phrase “wherein, upon induction of said inducible promoter, said meganuclease cleaves said vector at said recognition site” are unclear. The phrase is unclear in that the phrase provides method steps to a product claim. It would be remedial to replace the phrase “wherein, upon induction of said inducible promoter, said meganuclease cleaves said vector at said recognition site” with “wherein, upon induction of said inducible promoter, said meganuclease is capable of cleaving said vector at said recognition site”.
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 et al (Gene 331 (2003) 153-163) and further in view of Rodriguez-Garcia et al (Nucleic Acid Research; Vol. 33; 2005). This rejection was made in the Office action mailed 03/09/2026 and has been rewritten to address the amendment to the claims in the reply filed 07/09/2026.
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 replicate 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 wherein when the inducible promoter is induced, the meganuclease is capable of cleavage at the restriction site.
Herring teaches a two-plasmid system where one plasmid contains the I-Sce I gene and the other contains 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). Herring teaches the I-SceI meganuclease specifically cuts the donor plasmid, generating many copies of a linear fragment carrying the desired mutation in each cell (Page 157, Column 2). It would have been obvious to combine the I-Sce I gene and recognition sites on the same plasmid in order to only have to do one transformation instead of two and therefore, the I-SceI would cut the recognition site on the same plasmid. 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 and the recognition site within the same plasmid wherein I-SceI is a strong meganuclease 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 contains 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 1-12 and 16-18 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), has been maintained and rewritten as stated above.
Applicant’s arguments filed 07/09/2026 have been fully considered but they are not found persuasive.
Applicant argues that claim 1 has been amended to recite the function cooperation of the previously recited meganuclease gene, meganuclease restriction site, and inducible promoter on the claimed vector. Applicant continues to argue that claim 1 requires a single vector configured so that induction of the promoter causes expression of the vector-encoded meganuclease and cleavage of that same vector at the vector’s meganuclease recognition site.
However, the recited amendment to claim 1 raises indefiniteness under 35 U.S.C. 112(b), as stated above, due to the amendment imposing a method step into a product claim. It was there for interpreted, in the interest of compact prosecution, as “wherein, upon induction of said inducible promoter, said meganuclease is capable of cleaving said vector at said recognition site”. In addition, Herring teaches a two-plasmid system where one plasmid contains the I-Sce I gene and the other contains 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). Herring teaches the I-SceI meganuclease specifically cuts the donor plasmid, generating many copies of a linear fragment carrying the desired mutation in each cell (Page 157, Column 2). It would have been obvious to combine the I-Sce I gene and recognition sites on the same plasmid in order to only have to do one transformation instead of two and therefore, the I-SceI would cut the recognition site on the same plasmid. The inducible promoter, as taught by Rodriguez-Garcia, teaches that the use of a promoter is 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). Therefore, if the tcp830 promoter was linked to the I-SceI meganuclease, then the meganuclease would only perform its function of cleavage at the recognition site when it was induced. 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 and the recognition site within the same plasmid wherein I-SceI is a strong meganuclease 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.
Applicant argues 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 teachings of Biryukova does not teach a vector for the purpose of genome manipulation and instead “for production of useful metabolites” on Page 26. Applicant applies additional limitations out of context from the Biryukova prior art reference. Specifically, that the plasmid designed in Biryukova is not the instrument of editing but instead a replicative “maintenance and expression tool”.
However, where this quote is stated is specific to a paragraph describing other industrial applications of the plasmids designed and discussed within the prior art and not that the “production of useful metabolites” would be the only application of the invention described. The current claim limitations do not require only “genome editing”. The phrase “A vector for manipulation of a genome of a bacterium” also encompasses the increase or decrease of expression of genes within the genome of the bacterium, not necessarily the actual knock out or knock in of a gene within the genome of a bacterium.
Applicant argues that Biryukova does not disclose a vector-encoded meganuclease, a recognition site of that meganuclease on the same vector, or inducible cleavage of the vector by that encoded meganuclease. Applicant continues that Herring does not remedy the deficiencies of Biryukova. Applicant then argues that Sanchez-Romero or Rodriguez-Garcia do not remedy the deficiencies of Herring when teaching the meganuclease and recognition site on the same vector. Further, Applicant argues that Rodriguez-Garcia shows that an inducible promoter was known but does not teach or suggest the amended single-vector counter-selection configuration in which induction of the promoter causes a vector-encoded meganuclease to cleave the vector at its own recognition site.
However, 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 its own recognition site, specifically it would have been obvious to combine the I-Sce I gene and recognition sites on the same plasmid in order to only have to do one transformation instead of two and therefore, the I-SceI would cut the recognition site on the same plasmid. Rodriguez-Garcia teaches that the inducible promoter, the tcp830 promoter used with resistance marker genes, is a promoter induced by anhydrotetracycline for the purpose of increased expression when activated. 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 claimed invention requires “an origin of replication, wherein said origin of replication is not capable of inducing replication of said vector in said bacterium”. Applicant argues that Biryukova does not teach the origin of replication and Sanchez-Romero does not cure that deficiency. Specifically, the applicant argues that the fact that plasmid replication can be host-range dependent does not teach or suggest selecting an origin of replication that is not capable of inducing replication of the vector in the bacterium nor does it provide a reasoned basis to replace Biryukova’s RSF1010 oriV/rep system, which is used for broad host range replication and plasmid maintenance, with a non-replicative origin in the target bacterium.
However, the “origin of replication that is not capable of inducing replication of the plasmid within the bacteria” can also be referred to as a conditional origin of replication within a specific genus of bacterium. Wherein the origin of replication could be constitutively active in one genus of bacteria but not in another. When a conditional origin of replication is used within a plasmid delivered to a genus of bacteria where it is not constitutively active, the origin of replication can only be active in certain situations. 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). Therefore, the oriV origin of replication used within the plasmid of Biryukova would not have been constitutively active in E. coli bacteria that it would have been delivered to.
Therefore, Applicant’s arguments have not been found persuasive.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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 ALEXANDRA ROSE LIPPOLIS whose telephone number is (703)756-5450. The examiner can normally be reached Monday-Friday, 8:00am to 5:00pm EST.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637
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