Detailed Action
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-15, 17 and 19-21 are pending.
Claims 1-15 are withdrawn.
Claims 17 and 19-21 are examined.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 6/26/2025 has been entered.
Claim objections
Claim 20 is objected to because of the following informalities: in line 3, Applicant appears to have inadvertently included the word “or” after the verb “transfecting”. Appropriate correction is required.
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.
Claims 17 and 19-21 remain rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang et al. Genome Biology. 18:35. 2017) in view of Cigan (US 2015/0082478 A1).
Due to Applicant's amendment of the claims, the rejection was modified from the rejection set forth in the Office action mailed 3/26/2025, as applied to claims 17 and 19-21.
Claim 17 requires a method of editing a rice plant or cell by:
Introducing a first nucleic acid construct, a donor DNA element comprising a nucleic acid construct and editing a target gene of the plant with an efficiency of at least 12% wherein the nucleic acid construct has a structure of:
an expression cassette of a nucleic acid construct “Formula I” comprising in this order:
nothing or a nucleotide sequence
a first DSB sequence
a first homologous sequence
a target DNA sequence
a second homologous sequence
a second DSB sequence
nothing or a nucleotide sequence
the “first nucleic acid construct”, “Formula I’”
a promoter,
a coding sequence of Cas9 protein and
a terminator, in that order;
wherein each of the DSB sequences independently contains a cleavage site itself or forms a cleavage site after the nucleic acid construct is integrated into the target site by NHEJ. Both DSB sequences are each 10-50bp.
Claim 19 requires the method of claim 17 wherein the method further comprises a method for preparing a transgenic plant cell, comprising:
introducing a reagent combination, the reagent combination comprises:
a first nucleic acid construct “Formula I’” in this order:
a promoter,
a coding sequence of Cas9 protein and
a terminator, in that order;
a donor DNA element comprising a second nucleic acid construct “Formula II” comprising in this order:
a promoter
a coding sequence of gRNA
nothing or a transcription termination sequence and
an expression cassette of a nucleic acid construct “Formula I” comprising in this order:
nothing or a nucleotide sequence
a first DSB sequence
a first homologous sequence
a target DNA sequence
a second homologous sequence
a second DSB sequence
nothing or a nucleotide sequence
wherein the nucleic acid construct having a structure as shown in Formula I and a chromosome in the plant cell undergo site-directed knock-in or replacement to prepare the transgenic cell.
Claim 20 is drawn to the method of claim 17 wherein the nucleic acid construct of Formula I is introduced into the 3’ end of genes ACT1 and GST1 in rice.
Claim 21 is a method for preparing a transgenic plant by regenerating a plant made by the method of claim 19.
Zhang teaches a method of gene editing a cell using a nucleic acid construct comprising a DSB (a sgRNA1-PAM sequence that will guide Cas9 to create a DSB), a homologous sequence of a target gene which is used to knock in pD-mCherry, followed by another homologous sequence of the target gene (Wpre) (“Puro (663 bp) and Wpre (592 bp) serve as left and right HA [homologous arms], respectively”), followed by another DSB sequence (a sgRNA1-PAM that will guide Cas9 to create a DSB) (Figure 1 caption; page 2, right column, paragraph 2). Zhang teaches a nucleic acid construct encoding a Cas9 protein (page 14, paragraph bridging left and right columns). Zhang teaches co-transfection with these sequences (Figure 1 caption). Zhang teaches using EF1 promoter (Figure 1 caption). Zhang does not explicitly teach a terminator sequence; however, one of ordinary skill in the art would understand that a terminator must necessarily be used for expressing a protein such as a Cas9, for example, Zhang teaches use of a stop codon. Zhang teaches, “the double stranded break (DSB) is by Cas9/sgCTNNB1 39 bp before the stop codon”, i.e., the sequence is between 1-50bp (Figure 3 caption). Zhang teaches an efficiency of 14% at CTNNB1 and of 20-30% with the use of Nocodazole and CCND1 (page 13, paragraph bridging left and right columns).
Zhang does not teach the method of gene editing applied to a rice cell.
Cigan teaches a method of gene editing in a plant cell by introducing donor templates with homology arms at the site of a genomic double strand break with a Cas endonuclease introduced to the cell (Example 39, [0844]). Cigan teaches the plant is a rice plant (paragraph 24).
At the time of filing, it would have been prima facie obvious to one of ordinary skill in the art to combine the teachings of Cigan with the teachings of Zhang because it would be expected that expressing the nucleic acid constructs of Cigan would edit a gene in a plant cell with an efficiency of 14% or more. One would have been motivated to edit a gene of a plant cell with the constructs of Zhang in order to modify the traits of the plant. Therefore, the claimed invention is obvious over Zhang in view of Cigan.
Applicant’s arguments regarding rejection under 35 USC 103
Applicant's arguments filed 3/13/2025 have been fully considered but they are not persuasive.
Applicant argues that there would have been no reasonable expectation of success as the results of the claimed methods are unexpectedly superior to any of the art methods. Applicant argues that the achievement of Zhang having 14% and 20-30% efficiency as reading on the at least 12% efficiency claimed is not evidence of obviousness. Applicant argues that rice plants have lower than 2% efficiency with HDR DNA repair. HDR is more difficult in plants because of the rigid cell wall complicating delivery of genetic material and reducing efficiency. Therefore, precise gene editing via HDR is more difficult in plants than animals and the teaching of efficiency of Zhang would not be expected by applying teaching of Zhang with an HDR method in a plant. Applicant argues that the method of Zhang achieves a 4-fold improvement in HDR efficiency in 293T cells, referencing Figure 1d of Zhang which shows an increase from 5% to 20%, i.e. only a 4-fold increase, whereas the present invention achieves an editing efficiency of 12% in plant cells which is a 6-fold increase from the traditional method.
This argument has been fully considered but is not persuasive. While Applicant argues that the lack of HDR methods for plants teaching relative efficiency, there is nothing other than the limitation of applying the claimed method to a rice plant that differentiates the teachings of the prior art from the instantly claimed invention. While Applicant argues that the improvement of efficiency to 12% would not have been expected, it appears that applying the teachings of Zhang to edit a cell with the teachings of Cigan, which teaches modifying a plant cell, would result in at least the claimed amount of efficiency. I.e., it is not clear that there are any requirements of the instantly claimed limitation that differentiate applying the method of Zhang to a plant cell in view of the teachings of Cigan.
Applicant argues that the traditional methods in IPSCs had already achieved 10% HDR efficiency. Therefore, Zhang achieving 14% was still only a modest improvement and based more on the cell type (IPSCs) rather than Zhang’s editing system.
This argument has been fully considered but it is not persuasive. While Zhang is not drawn to gene editing a rice plant cell, IPSCs can be derived from plant cells. Further, it is not clear that applying the method of Zhang in view of Cigan to a rice plant cell would not have resulted in the claimed efficiency, i.e., there is no difference between the instantly claimed method from applying the method of Zhang to a rice plant cell in view of Cigan.
Applicant argues that Zhang’s 20-30% efficiency was achieved by cell cycle regulators to optimize cell cycle conditions, therefore this does not support Zhang’s method achieving high HDR efficiency.
This argument has been fully considered but it is not persuasive. It is not clear that applying the method of Zhang in view of Cigan to a rice plant cell would not have resulted in the claimed efficiency, i.e., there is no difference between the instantly claimed method from applying the method of Zhang to a rice plant cell in view of Cigan.
Conclusion
Claims 17 and 19-21 remain rejected.
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/DAVID R BYRNES/Examiner, Art Unit 1662