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 .
Status of Claims
The preliminary amendment filed on 07/22/2025 amended claims 3, 5, 9, 11, 13, 21, 26-27, 29-30, 33, 39, 57, and 60, and cancelled claims 4, 6-8, 10, 12, 14-20, 22-25, 28, 34-38, 40-56, and 61-87. Claims 1-3, 5, 9, 11, 13, 21, 26-27, 29-33, 39, and 57-60 are pending and will be examined on the merits.
Priority
Provisional application 63/558,983 is acknowledged as disclosing the claimed invention and the effective filing date is 02/28/2024.
Information Disclosure Statement
The Information Disclosure Statement filed on 05/04/2026 has been considered. Signed copies are enclosed.
Claim Objections
Claim 30 is objected to because of the following informalities: Claim 30 recites that the donor vectors are configured to incorporate target DNA inserts from "(i) Golden Gate vectors, GoldenBraid plasmids, Mobius Level-2 plasmids, and Loop pEven plasmids; (ii) MoClo plasmids; or (iii) either (i) or (ii)." (iii) is redundant, as the "or" present in the claim already indicates that either (i) or (ii) is required. Appropriate correction is required.
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.
Claims 5, 27, and 60 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.
Claims 5 and 60 recite the term “a recombineering system”. This term is not properly defined by the claim or the instant specification, and one of ordinary skill in the art would not reasonably know what “a recombineering system” comprises. For the purposes of applying prior art, Examiner is interpreting “a recombineering system” as any recombinase.
Claim 27 recites “a Donor I vector or a Donor II vector.” This term is not properly defined by the claim or the instant specification and is not a term commonly used in the art. It is unclear what limitations are imposed by the term “a Donor I vector or a Donor II vector.” For the purposes of applying prior art, Examiner will be interpreting “a Donor I vector” as any first donor vector, and “a Donor II vector” as any second donor vector, as indicated in the specification, paragraph [0098].
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1, 2, 5, 21, 27, 29-33, 39, and 57-60 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Collier et al., The Plant Journal, (2018), 95, 573–583.
The instant claims are drawn to a gene stacking system comprising at least one donor vector, an acceptor vector configured to contain up to about 300 kb of target DNA inserts, and a first bacterial cell comprising the first acceptor vector. Claim 31, an alternate embodiment of claim 1, recites the same system, but comprises an additional donor vector, and a recombinase in the first bacterial cell. Claim 58, a third embodiment, recites the at least one donor vector comprises at least one target DNA insert flanked by recombination donor sites, the at least one acceptor vector comprises recombination acceptor sites compatible with the donor sites, and an engineered bacterial cell comprising the acceptor vector and at least one recombinase.
Collier et al. teach a gene stacking system known as GAANTRY, comprising four plasmid vectors and a recipient bacterial strain, wherein two of the plasmid vectors are donor vectors (“P and B ‘donor vectors’”, page 574, right column). Collier et al. teach that the acceptor vector is a pRi virulence plasmid, referred to as ArPORT1, which is configured to receive at least 28.5 kb of DNA, and thus is configured to accept up to about 300kb. Therefore, Collier et al. anticipate instant claim 1 and 2. Collier et al. further teach that the cells comprise the recombinases, ParA and A118. Therefore, Collier et al. teach a recombineering system, and anticipate claim 5, as well as claims 31, 58, and 60. Collier et al. teach that these recombinases are expressed transiently, and are thus inducible, teaching instant claim 59. The donor vectors of Collier et al. carry recombinase recognition sites (attB and attP) flanking target DNA inserts, as well as the sacB gene, and carry target DNA inserts that will be inserted into the acceptor vector, teaching claims 21, 27, 32, and 39. Collier et al. teach that integration can be repeated by alternating vectors, teaching instant claim 33. Collier et al. teach that these vectors may be manipulated in E. coli strains using any standard cloning technique for insertion of the target sequences, and the cargo can be from any cloning technology, including Golden Gate and Golden Braid based techniques, which are Type IIS restriction enzyme-based cloning technologies, therefore teaching claims 29, 30, and 57. Therefore, Collier et al. teach instant claims 1, 2, 5, 21, 27, 29-33, 39, and 57-60.
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.
Claim(s) 1-3, 5, 9, 13, 21, 26, 27, 29-33, 39, and 57-60 is/are rejected under 35 U.S.C. 103 as being unpatentable over Collier et al. in view of Brumos et al., The Plant Cell, Vol. 32: 100–122, (2020).
The instant claims are drawn to a gene stacking system comprising at least one donor vector, an acceptor vector configured to contain up to about 300 kb of target DNA inserts, and a first bacterial cell comprising the first acceptor vector. Claim 31, an alternate embodiment of claim 1, recites the same system, but comprises an additional donor vector, and a recombinase in the first bacterial cell. Claim 58, a third embodiment, recites the at least one donor vector comprises at least one target DNA insert flanked by recombination donor sites, the at least one acceptor vector comprises recombination acceptor sites compatible with the donor sites, and an engineered bacterial cell comprising the acceptor vector and at least one recombinase.
Collier et al. teach a gene stacking system known as GAANTRY, comprising four plasmid vectors and a recipient bacterial strain, wherein two of the plasmid vectors are donor vectors (“P and B ‘donor vectors’”, page 574, right column). Collier et al. teach that the acceptor vector is a pRi virulence plasmid, referred to as ArPORT1, which is configured to receive at least 28.5 kb of DNA, and thus is configured to accept up to about 300kb. Therefore, Collier et al. teaches instant claim 1 and 2. Collier et al. further teach that the cells comprise the recombinases, ParA and A118. Therefore, Collier et al. teach a recombineering system, and teach claim 5, as well as claims 31, 58, and 60. Collier et al. teach that these recombinases are expressed transiently, and are thus inducible, teaching instant claim 59. The donor vectors of Collier et al. carry recombinase recognition sites (attB and attP) flanking target DNA inserts, as well as the sacB gene, and carry target DNA inserts that will be inserted into the acceptor vector, teaching claims 21, 27, 32, and 39. Collier et al. teach that integration can be repeated by alternating vectors, teaching instant claim 33. Collier et al. teach that these vectors may be manipulated in E. coli strains using any standard cloning technique for insertion of the target sequences, and the cargo can be from any cloning technology, including Golden Gate and Golden Braid based techniques, which are Type IIS restriction enzyme-based cloning technologies, therefore teaching claims 29, 30, and 57.
Collier et al. do not explicitly teach the bacterial cell comprising an arabinose-inducible-FLP recombinase, the first bacterial cell derived from an E. coli SW105 strain, or compatibility with an Agrobacterium tumefaciens GV3101 strain, as the GAANTRY system is engineered for A. rhizogenes.
Brumos et al. teach an improved recombineering toolset for plants, comprising a BAC-based vector known as pYLTAC17 comprising an exchange cassette of FRT2-SacB-FRT5, or a SacB gene flanked by FLP-recombinase sites (see page 107, left col.). The instant specification teaches, in paragraph [0010], that using a transformable BAC backbone brings the upper limit of DNA cargo size to 300 kb. Therefore, Brumos et al. teach an acceptor vector configured to hold up to 300kb of DNA inserts, as recited in instant claims 1, 31, and 58. Brumos et al. teach this vector in an E. coli SW105 strain, which contains an arabinose-inducible FLP recombinase, teaching the limitations of claims 9 and 13 (see page 102, right col.). The FRT2-SacB-FRT5 cassette teaches the limitations of instant claims 21. The system is taught to be compatible with A. tumefaciens strain UIA143 pMP90 (See page 118, section “Plant Transformation and Fluorescence Analysis”), known in the art to be derived from a GV3101 strain (as evidenced by Hamilton, 1997). Therefore, the system is compatible with a GV3101 strain of Agrobacterium, teaching the limitations of instant claim 26.
Taking the teachings of both Collier et al. and Brumos et al. together, it would be obvious to one of ordinary skill in the art to arrive at the claimed invention before the effective filing date. Collier et al. disclose a system, GAANTRY, very similar to the claimed system. It would be obvious to one of ordinary skill in the art, before the effective filing date of the invention, to modify the GAANTRY system and the ArPORT1 plasmid to a pYLTAC17-based cloning system, similar to that disclosed in Brumos et al. One would be motivated to do so in order to expand the species the system can work with beyond A. rhizogenes, which the ArPORT1 vector is limited to, and to be able to include more DNA inserts, as the pYLTAC17 vector is a BAC-based vector designed for the generation of large-insert DNA constructs. Brumos et al. disclose, on page 107, left col., that the use of pYLTAC17 allowed for an increase in the number of transformants and the integrity of the DNA inserts when using the pYLTAC17 backbone, as compared to other vectors. Therefore, it would be obvious to one of ordinary skill in the art to modify the acceptor vector of Brumos et al., for use in the GAANTRY system. One would have reasonable expectation of success, as Brumos et al. show that the pYLTAC17 plasmid can be genetically manipulated to contain recombination sites and still have better transformation efficiency and integrity compared to other plasmids. Therefore, it is obvious to modify the system of Collier et al. with a BAC-based vector, allowing for DNA inserts of up to 300 kb.
It is then obvious to modify the system of Collier et al. to manipulate the donors of GAANTRY in an E. coli SW105 strain, as this strain contains an arabinose-inducible FLP recombinase, and the pYLTAC17 vector contains FLP recombinase specific sites. As recited in Collier et al., page 574, right col., the GAANTRY donors can be manipulated by any standard technique in E. coli and can receive cargo from any cloning technology, including Golden Gate, GoldenBraid, or their variants, Loop, Mobius, and MoClo, indicating that the limiting . Therefore, it would be obvious to modify the donor plasmids to use FLP recombinase in place of ParA recombinase, allowing for the pYLTAC17 vector to receive the cargo from the GAANTRY donors without further manipulation. Additionally, the arabinose-inducible FLP recombinase provides an alternate means of transient expression of the recombinase, and allows for the assembly of the construct to occur in E. coli, an easier organism to genetically manipulate than Agrobacterium. Additionally, as the pYLTAC17 vector is compatible with the GV3101 strain of A. tumefaciens, the system of Collier et al. in view of Brumos et al. would also be compatible with the GV3101 strain of A. tumefaciens, allowing for the GAANTRY system to be efficiently adapted for use in A. tumefaciens. One would be motivated to use the GAANTRY system in A. tumefaciens over A. rhizogenes as the two different species can target different plants, allowing for broader genetic manipulation.
Finally, it would be obvious to one of ordinary skill in the art to perform the method of Collier et al. in view of Brumos et al. with two different sets of target DNA inserts in order to target two different genetic changes in a plant. One would be motivated to do so if the changes desired to be made were larger than 300 kb each, or if the genes were for some reason incompatible with each other, or if the target genes were intended for a second target plant. In that case, it would be obvious to have a second bacterial cell comprising a second acceptor vector, as in instant claim 3, so that each cell can contain the intended DNA. Therefore, claim 3 is obvious over Collier et al. in view of Brumos et al., as well as claims 1, 2, 5, 9, 13, 21, 26-27, 29-33, 39, and 57-60.
Claim(s) 1-3, 5, 9, 11, 13, 21, 26, 27, 29-33, 39, and 57-60 is/are rejected under 35 U.S.C. 103 as being unpatentable over Collier et al. in view of Brumos et al. as applied to claims 1-3, 5, 9, 13, 21, 26, 27, 29-33, 39, and 57-60 above, and further in view of Xu and Brown, BMC Biotechnology (2016) 16:13.
The teachings of Collier et al. in view of Brumos et al. regarding claims 1-3, 5, 9, 13, 21, 26, 27, 29-33, 39, and 57-60 are recited above. Collier et al. and Brumos et al. do not teach the second recombinase as an inducible PhiC31 recombinase.
However, as taught by Xu and Brown, the PhiC31 recombinase is in the same family of recombinases as A118, the equivalent recombinase in the GAANTRY system. As recited on page 8, left col. of Xu and Brown, the PhiC31 recombinase activity is greater than the A118 recombinase activity, the recombinase used in Collier et al. Therefore, one of ordinary skill in the art of serine recombinases would be motivated to use the phiC31 recombinase over the A118 recombinase used in the GAANTRY system. One would have reasonable expectation of success, as Xu and Brown show that the two are equivalents in the art and can both work in exogenous systems. Moreover, the recombinases of Xu and Brown are inducible, allowing recombination to only take place when expression of recombinase is induced. Similarly, Collier et al. use suicide vectors encoding the recombinase A118 to control recombinase expression, so that the recombinase is only initially expressed upon transduction in the bacterium, and Brumos et al. use an inducible system for FLP recombinase, so that the recombinase is only expressed when induced. Taken together, one would be motivated to use the inducible recombinase phiC31 of Xu and Brown with the inducible FLP recombinase of Brumos et al., recited above, so that the two inducible recombinases can function in tandem in the gene-stacking system and suicide vectors do not have to be re-transduced each time they are needed. Therefore, claim 11 is obvious over Collier et al. in view of Brumos et al. and further in view of Xu and Brown.
Conclusion
No claim is allowed.
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/AMELIA STEPHENS/ Examiner, Art Unit 1645
/ANNE M. GUSSOW/ Supervisory Patent Examiner, Art Unit 1683
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