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 .
Election/Restrictions
Applicant’s election without traverse of group V in the reply filed on May 13, 2026 is acknowledged.
Group V as set forth in the restriction requirement mailed January 14, 2026 was directed to claims 9-11, drawn to the method of claim 1 wherein the Cas9 endonuclease protein is cotransfected with one or more plasmids encoding one or more exonucleases. Claim 1 linked inventions I-IX.
Applicant maintains that following the entry of the proposed claim amendments, claims 1-18 should read upon the elected group, group V.
Applicant’s proposal is partially persuasive. Because claims 1-8 and 11-17 as currently amended are now directed to the subject matter of Group V, claims 1-8 and 11-17 are examined together on the merits herein. However, because claim 18 is directed to subject matter of group X, claim 18, and new claim 19 which depends therefrom, are withdrawn.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 13 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 13 fails to further limit the subject matter of claim 12 because claim 12 is limited to specific to crop species, whereas claim 13 is directed to a plant genus. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim 14 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 14 fails to further limit the subject matter of claim 12 because claim 12 does not recite Arabidopsis thaliana as a member of the group of alternative plant species. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
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) 1-8 and 11-17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yang et al. (U.S. Patent Application Publication No. 2015/0067922, published Mar. 5, 2015) in view of Certo et al. (Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nat. Methods. 2012 Oct;9(10):973-5. Epub 2012 Sep 2), Samuel et al. (U.S. Patent Number 9,187,755, issued Nov. 17, 2015), Doudna et al. (U.S. Patent Application Publication No. 2014/0068797, published Mar. 6, 2014), Chugh et al. (Cell-penetrating peptides: Nanocarrier for macromolecule delivery in living cells. IUBMB Life. 2010 Mar;62(3):183-93) and Martin-Ortigosa et al. (U.S. Patent Application Publication No. 2014/0096284, published Apr. 3, 2014).
Claim 1 is drawn to a method for targeted genetic modification of a plant genome without inserting exogenous genetic material into the genome, the method comprising: co-transfecting a plant cell with one or more plasmids encoding a TREX exonuclease, a purified Cas9 endonuclease protein, and a guide RNA using biolistic or protoplast transformation, wherein said TREX exonuclease is expressed in the plant cell and said Cas9 endonuclease introduces one or more double stranded DNA breaks (DSB) in the genome to produce a plant cell or cells having a detectable targeted genomic modification without the presence of any exogenous Cas9 genetic material in the plant genome.
Claim 2 is drawn to the method of claim 1, wherein said one or more DSBs are repaired by non-homologous end joining (NHEJ).
Claim 3 is drawn to the method of claim 1, wherein introduction of one or more DSBs in the genome is followed by repair of the one or more DSBs through a homologous recombination mechanism.
Claim 4 is drawn to the method of claim 1, wherein the Cas9 endonuclease further comprises one or more subcellular localization domains.
Claim 5 is drawn to the method of claim 4, wherein the one or more subcellular localization domains comprise an SV40 nuclear localization signal, an acidic M9 domain of hnRNPA1, a PY-NLS motif signal, a mitochondrial targeting signal, or a chloroplast targeting signal.
Claim 6 is drawn to the method of claim 1, wherein the Cas9 endonuclease further comprises one or more cell penetrating peptide domains (CPPs).
Claim 7 is drawn to the method of claim 6, wherein said one or more CPPs comprise a transactivating transcriptional activator (Tat) peptide.
Claim 8 is drawn to the method of claim 6, wherein said one or more CPPs comprise a Pep-1 CPP domain.
Claim 11 is drawn to the method of claim 1, wherein the member of the TREX exonuclease family is TREX2.
Claim 12 is drawn to the method of claim 1, wherein said plant cell is from a crop species of alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, or wheat.
Claim 13 is drawn to the method of claim 12, wherein said plant cell is from the genus Nicotiana.
Claim 14 is drawn to the method of claim 12, wherein said plant cell is from the species Arabidopsis thaliana.
Claim 15 is drawn to the method of claim 1, wherein transfection is effected through delivery of said purified Cas9 endonuclease protein into isolated plant protoplasts.
Claim 16 is drawn to the method of claim 1, wherein transfection is effected through delivery of said purified Cas9 endonuclease protein by biolistic transformation.
Claim 17 is drawn to the method of claim 1, further comprising regenerating the plant cell or cells having the detectable targeted genomic modification into a plant.
Yang et al. teach a method for targeted genetic modification of a plant genome, the method comprising: (i) providing a rice or potato plant cell protoplast that comprises an endogenous gene to be modified; (ii) providing a nucleic acid encoding a Cas9 endonuclease protein that further comprises a nuclear localization signal and a guide RNA targeted to the endogenous gene; and (iii) transfecting or transforming the plant cell with said nucleic acid encoding a Cas9 endonuclease protein and a guide RNA, such that said Cas9 endonuclease introduces one or more double stranded DNA breaks (DSB) in the genome to produce a plant cell or cells having a detectable targeted genomic modification produced by cellular processes that include non-homologous end joining (NHEJ) and homologous recombination (HR) (paragraphs [0123] – [0174]). Yang et al. also teach the use of plasmids encoding proteins, as well as biolistic and protoplast transformation methods (paragraphs [0078], [0105], [0106], [0116]). Yang et al. additionally teach Nicotiana (tobacco) and Arabidopsis thaliana as among the plant species that can be genetically modified by their methods (paragraph [0050], [0103]). Yang et al. further teach that their methods can be practiced using non-transgenic approaches, i.e. approaches that would result in the production of plant cells that have a detectable targeted genomic modification without the presence of any exogenous Cas9 genetic material in the plant genome, which approaches should significantly improve public acceptance of genetically engineered plants (paragraphs [0024], [0122]).
Yang et al. do not teach co-transfecting a plant cell with one or more plasmids encoding a TREX exonuclease, a purified Cas9 endonuclease protein, and a guide RNA using biolistic or protoplast transformation.
Certo et al. teach methods for targeted genetic modification of animal genomes by coexpressing a TREX2 exonuclease and a designer endonuclease that is an I-SceI endonuclease, a ZFN endonuclease, or a TALEN endonuclease. The coexpression of TREX2 exonuclease and a designer endonuclease enhanced genetic modification as compared to expression of a designer endonuclease alone.
Samuel et al. teach a method comprising providing a plant cell; coating a nanoparticle with a sequence-specific nuclease (SSN); placing the plant cell and the coated nanoparticle in contact with each other; and allowing uptake of the nanoparticle and the SSN into the plant cell, including a method further comprising selecting the plant cell after uptake of the SSN into the plant cell, including wherein the selected cell is a regenerable cell, and including a method further comprising regenerating a plant from the regenerable cell (claims 1-13). The sequence-specific nuclease of Samuel et al. can be in the form of a purified protein (column 17 line 53-61; column 19 lines 3-5). The sequence-specific nuclease of Samuel et al. can induce one or more double stranded DNA breaks (DSB) in the genome (column 2 line 48 to column 3 line 7). The method of Samuel et al. can modify an endogenous gene of the plant cell without inserting exogenous genetic material (column 17 line 53-61). Samuel et al. also teach that when a purified sequence-specific nuclease is delivered into target plant cells, surgically specific mutations and gene knock-outs can be induced via non-homologous end joining (NHEJ), which in turn can produce a non-transgenic genetically modified plant that would bypass restrictions on transgenic crops, making the process of targeted gene editing possible without requiring a transgenic approach (column 17 line 53-61).
Doudna et al. teach a method of site-specific modification of a target DNA in a plant cell, the method comprising: contacting the target DNA with: (i) a DNA-targeting RNA, or a DNA polynucleotide encoding the same, wherein the DNA-targeting RNA comprises: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) a site-directed modifying Cas9 polypeptide, or a polynucleotide encoding the same, wherein the site-directed modifying polypeptide comprises: (a) an RNA-binding portion that interacts with the DNA-targeting RNA; and (b) an activity portion that exhibits site-directed nuclease enzymatic activity that introduces a double strand break in the target DNA, wherein the contacting occurs under conditions that are permissive for nonhomologous end joining or homology-directed repair (claims 64, 68, 69, 70, 73, 76, 77, 78). Doudna et al. also teach that the site-directed modifying polypeptide may be provided to cells as a polypeptide, including as a polypeptide fused to a polypeptide permeant domain known to promote uptake by the cell, such as penetratin, the permeant peptide that comprises the HIV-1 tat basic region amino acid sequence, and permeant domains that include poly-arginine motifs (paragraphs [0287] - [0288]). Doudna et al. further teach that the site-directed modifying polypeptide may be produced in vitro or by eukaryotic cells or by prokaryotic cells, and may be further processed by unfolding, and may be further refolded, using methods known in the art (paragraph [0289]). Doudna et al. also teach the fusion of a Cas9 polypeptide with a heterologous sequence that provides for subcellular localization of the polypeptide, such as a nuclear localization signal for targeting to the nucleus; a mitochondrial localization signal for targeting to the mitochondria, and a chloroplast localization signal for targeting to a chloroplast (paragraph [0241]).
Chugh et al. teach the use of cell-penetrating peptides to deliver proteins and nucleic acids to plant protoplasts, including Tat and Pep-1 (page 189 column 1 through column 2 first full paragraph).
Martin-Ortigosa et al. teach the transfection of plant cells that is effected through the delivery of purified proteins, DNA, and other chemicals, by biolistic transformation (Table 1; Examples 1-V).
Given the teachings of Yang et al. that an endogenous gene in a plant genome can be targeted and modified by a Cas9 endonuclease protein expressed from a nucleic acid encoding the Cas9 endonuclease protein that has been introduced into a plant cell protoplast, given the teachings of Yang et al. that plasmids encoding proteins, and biolistic and protoplast transformation methods, can be used in methods for targeted genetic modification of a plant genome, given the teachings of Yang et al. that their methods can be practiced using non-transgenic approaches, i.e. approaches that would result in the production of plant cells that have a detectable targeted genomic modification without the presence of any exogenous Cas9 genetic material in the plant genome, which approaches should significantly improve public acceptance of genetically engineered plants, given the teachings of Certo et al. that the coexpression in animal cells of a TREX2 exonuclease and a designer endonuclease that is an I-SceI endonuclease, a ZFN endonuclease, or a TALEN endonuclease enhances genetic modification as compared to expression of a designer endonuclease alone, given the teachings of Doudna et al. that an endogenous gene in a plant genome can be targeted and modified by a Cas9 endonuclease protein expressed from a nucleic acid encoding the Cas9 endonuclease protein that has been introduced into a plant cell or by a Cas9 endonuclease protein that has been introduced into a plant cell, given the teachings of Samuel et al. that delivery of a purified sequence-specific nuclease into target plant cells can result in the production of surgically specific mutations and gene knock-outs via non-homologous end joining (NHEJ) to produce a non-transgenic genetically modified plant, given the teachings of Chugh et al. that cell-penetrating peptides can be used to deliver proteins and nucleic acids to plant protoplasts, and given the teachings of Martin-Ortigosa et al. that transfection of plant cells can be effected through the purified proteins, DNA, and other chemicals, by biolistic transformation, it would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to co-transfect a plant cell with one or more plasmids encoding a TREX2 exonuclease, a purified Cas9 endonuclease protein, and a guide RNA, using biolistic or protoplast transformation, to produce a plant cell or cells having a detectable targeted genomic modification without the presence of any exogenous Cas9 genetic material in the plant genome.
One skilled in the art would have been motivated to do so in order to enhance the genetic modification of the plant cell by the Cas9 endonuclease, as compared to the genetic modification of the cell by the Cas9 endonuclease alone, to produce a plant cell or cells having a detectable targeted genomic modification without the presence of any exogenous Cas9 genetic material in the plant genome.
One skilled in the art would have had a reasonable expectation of success, given the success of Certo et al. in using aTREX2 exonuclease in combination with I-SceI, ZFN, and TALEN endonucleases in animal cells, and given the success of Yang et al. in modifying the genome of a plant cell using a Cas9 endonuclease.
One skilled in the art also would have been motivated to deliver a purified Cas9 endonuclease protein and a guide RNA to a plant cell in order to produce a non-transgenic genetically modified plant therefrom that would bypass restrictions on transgenic crops.
One skilled in the art would have had a reasonable expectation of success, given the success of Yang et al. in targeting and modifying an endogenous plant gene by expressing in a plant cell protoplast a Cas9 endonuclease protein from a nucleic acid that has been introduced into the cell, and given the success of others in introducing proteins and nucleic acids directly into plant cells and plant protoplasts.
One skilled in the art would have recognized that one or more DSBs introduced by the Cas9 endonuclease could be repaired by non-homologous end joining (NHEJ) or by a homologous recombination mechanism, since such cellular repair mechanisms were already known in the art, as evidenced by Yang et al., Samuel et al., and Doudna et al.
One skilled in the art would have recognized plant cells from crop species such as species within the genus Nicotiana, or the species Arabidopsis thaliana, are suitable species for targeted genetic modification, as well as for regeneration into a plant, since the prior art already recognized this, as evidenced by Yang et al. and Samuel et al.
One skilled in the art would have recognized the use of both biolistic and protoplast transformation methodologies as among a finite number of alternatives for delivering a purified protein and/or nucleic acid to a plant cell and/or a plant protoplast, since the prior art teaches that these methods are useful for this purpose.
One skilled in the art would have recognized that subcellular localization domains could be used to target a Cas9 endonuclease to a specific subcellular compartment where its activity is desired, since the prior art already recognized this, as evidenced by Yang et al. and Doudna et al.
One skilled in the art would have recognized that cell penetrating peptide domains (CPPs) could be used to facilitate the introduction of a purified protein into a plant cell, since the prior art already recognized this, as evidenced by Doudna et al. and Chugh et al.
Thus the claimed invention would have been prima facie obvious as a whole to one of ordinary skill in the art before the effective filing date of the claimed invention.
Remarks
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA E COLLINS whose telephone number is (571)272-0794. The examiner can normally be reached M-F 8:30 am - 5:00 pm.
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/CYNTHIA E COLLINS/Primary Examiner, Art Unit 1662
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