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 .
Priority
The instant application, filed on 03/13/2023 claims foreign priority to EP 20196437.6 filed on 09/16/2020 and benefit of priority to PCT/EP2021/075388 filed on 09/15/2021. Acknowledgement is made of Applicant’s claim for foreign priority under 35 U.S.C. 119(a)-(d) and domestic benefit under 35 U.S.C. 119(e). The Certified Copy of the Foreign Priority Application was received on 03/13/2023. Thus, the effective filing date of Claims 1-6 and 8-19 is 09/16/2020.
Status of Claims
Amendments dated 09/29/2025 have been entered.
Claim 7 is cancelled by Applicant.
Claims 15-19 are newly added.
Claims 1-6 and 8-19 are pending.
Claims 1-6 and 8-19 are examined herein.
The objections to the Drawings are withdrawn in view of Applicant’s amendments to the Drawings and Sequence Listing.
The objections to Claims 10 and 12 are withdrawn in view of Applicant’s amendments to the claims.
The rejections to Claims 2, 4-5, 7 and 12-14 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention are withdrawn in view of Applicant’s amendments to the claims and cancellation of Claim 7.
The rejection of Claim 7 on the ground of nonstatutory double patenting as being unpatentable over Claims 6-7, 11-12, and 15 of copending Application No. 18/026,102 (US 2023/0357783, filed 03/13/2023) in view of Bortesi et al. (EP 3392339 A1, published 10/24/2018) is withdrawn in view of Applicant’s cancellation of the claim.
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 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.
---The following rejections are modified from those set forth in the Office Action dated 05/02/2025 in view of Applicant’s amendments to the claims dated 09/29/2025 and addition of new Claims 15-19---
Claims 1-4, 6-11, 13-14, and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Bortesi et al. (EP 3392339 A1, published 10/24/2018) in view of Schubert et al. ("Fluorescently labeled tracrRNA provides efficient genome editing while allowing cellular microscopy and FACS analysis" ©2017) and Rukmana et al. (APL photonics 5.6, published 06/15/2020).
Regarding Claim 1, Bortesi et al. (herein referred to as Bortesi) teaches a method for altering the genome of an intact differentiated cell(s) without inserting exogenous genetic material into the genome comprising: (i) providing an intact differentiated cell that comprises an endogenous gene to be modified; (ii) providing a genome-modifying formulation comprising a pre-assembled ribonucleoprotein (RNP)-complex comprising a nucleic acid modifying protein and a ribonucleic acid; (iii) delivering said RNP-complex into the cell by using a physical delivery method; (iv) inducing one or more single or double stranded DNA breaks in the cell genome to produce a cell having a detectable targeted genomic modification without the presence of any exogenous genetic material in the cell genome (pg. 23, Claim 1), wherein the cell is a plant cell comprising a cell wall, in particular said plant cell is from a plant from the Solanaceae family, like Nicotiana tabacum and Nicotiana benthamiana from the genus Nicotiana (pg. 24, Claim 9), wherein said delivery method is abrasion and/or perforation or wherein said delivery method is a biolistic particle delivery method (pg. 24, Claim 11). Bortesi teaches that an embodiment of their invention includes physical delivery methods, such as perforation, wherein perforation of the RNP complex is performed with echo waves or a laser (paragraph 0011).
Regarding Claim 4, Bortesi teaches a method for altering the genome of an intact differentiated cell(s) without inserting exogenous genetic material into the genome comprising: (i) providing an intact differentiated cell that comprises an endogenous gene to be modified; (ii) providing a genome-modifying formulation comprising a pre-assembled ribonucleoprotein (RNP)-complex comprising a nucleic acid modifying protein and a ribonucleic acid; (iii) delivering said RNP-complex into the cell by using a physical delivery method; (iv) inducing one or more single or double stranded DNA breaks in the cell genome to produce a cell having a detectable targeted genomic modification without the presence of any exogenous genetic material in the cell genome (pg. 23, Claim 1), wherein the cell is a plant cell comprising a cell wall, in particular said plant cell is from a plant from the Solanaceae family, like Nicotiana tabacum and Nicotiana benthamiana from the genus Nicotiana (pg. 24, Claim 9), wherein said delivery method is abrasion and/or perforation (pg. 24, Claim 11). Bortesi teaches that plant cells useful in the methods of the invention also include plant cells in the form of leaves, stems, cotyledons, and pollen (pg. 5, paragraph 0019). Bortesi teaches that an embodiment of their invention includes physical delivery methods, such as perforation, wherein perforation of the RNP complex is performed with echo waves or a laser (paragraph 0011). Bortesi teaches that the RNP complex was added to a microcarrier and the leaf bits were placed there prior to delivery of the RNP complex through physical delivery methods (Example 1, paragraph 0075). Bortesi teaches in two working examples (Example 1 and Example 2) that the plant cells that had the RNP complex delivered to them were observed using fluorescence microscopy to determine whether or not the delivery of the RNP complex to the cells resulted in efficient DNA cleavage (Example 1, paragraphs 0075-0077; Example 2, paragraphs 0078-0079). Bortesi teaches in several working examples that plants cells which were identified to have undergone genome editing or modification due to the introduction of the RNP complex into said plant cell were subsequently regenerated into plants using various regeneration media (Example 5, paragraphs 0085-88; Example 8, paragraphs 0093-94; Example 9d, paragraphs 0100-101; Example 9F, paragraph 0103; Example 10b, paragraphs 0105-106).
However, Bortesi does not explicitly teach the limitations of Claims 1 and 4, wherein the ribonucleic acid is labeled with a visual marker or wherein the laser focal point is in the liquid adjacent to the cell wall and not directly in contact with the cell wall, the step of Claim 4 comprising layering the plant explant onto the RNP-complex formulation in a transparent support, or the limitation of Claim 6, wherein the laser irradiation conditions are first adjusted before delivering of the RNP-complex by the laser-assisted transfection method.
Regarding Claims 1 and 4, Rukmana et al. (herein referred to as Rukmana) teaches a method of using a laser-assisted transfection to create temporary pores in a Nicotiana tabacum plant cell membrane, allowing for the introduction of molecular like fluorescent nanoparticle (Abstract). Rukmana teaches that plant cells with a detectable fluorescence intensity were selected as successfully transfected (pg. 066104-8, last paragraph). In the context of photo-injection in plant cells, Rukmana teaches that placing the laser focal point adjacent to the plant cell wall in the culture medium (10 µm away from plant cell wall), without directly contacting it, allows for targeted molecular delivery without causing significant damage to cell wall (pg. 066104-7, Figure 8; pg. 066104-6, paragraph bridging left and right column). Additionally, Rukmana teaches that the laser is applied to the liquid medium through a filter; thus, the laser is also not directly in contact with plant cells or plant cell walls (pg. 066104-2, NIR fs laser photoinjection). Before the Nicotiana tabacum wild-type TBY-2 cell suspensions are photoinjected, Rukmana teaches that 100 microliters of the cell suspensions in each sample condition were placed in a glass-bottom dish (transparent support) and that the enzymatic pre-treatment did not remove the cell wall (pg. 5, right column, first full paragraph).
Regarding Claim 6, Rukmana teaches that the laser irradiation conditions are adjusted prior to delivery of the biomolecule through the laser assisted transfection method (pg. 066104-2, NIR fs laser photoinjection).
It would have been obvious to one of ordinary skill in the art to modify the method for altering the genome of a plant comprising introducing into the plant cell an RNP complex taught by Bortesi, wherein the RNP complexes are delivered to the cell through passive uptake of the RNP complex which is facilitated by creating local lesions in the cell wall by physical delivery methods including mechanical methods like perforation (e.g. with echo waves or laser) to have the laser focal point be focused on the culture medium directly adjacent to the cell wall and for the cell suspension and culture medium to be in a glass bottom dish, as taught by Rukmana, to allow for targeted molecular delivery without causing significant damage to cell wall (pg. 066104-7, Figure 8; pg. 066104-6, paragraph bridging left and right column) and the ability to view the photoinjected cell suspensions under a microscope (pg. 066104-3, Figure 3). Rukmana also teaches that applying the laser assisted transfection method in Nicotiana tabacum plant cells is expected to be successful when expanded to gene modifications in a plant system (pg. 066104-8, Conclusion). As such, Rukmana provides a strong motivation and high expectation of success to one of ordinary skill in the art to modify the method of Bortesi to include adjusting the laser focal point be focused on the culture medium directly adjacent to the cell wall and for the cell suspension and culture medium to be in a glass bottom dish prior to photoinjection, as taught by Rukmana.
However, Rukmana does not explicitly teach the limitations of Claims 1 and 4, wherein the ribonucleic acid is labeled with a visual marker.
Regarding Claims 1 and 4, Schubert et al. teaches that rather than fluorescently labeling a Cas9 protein (which increases the cargo size of the RNP and may reduce delivery efficiencies), a more practical, effective approach for RNP detection is to fluorescently label the chemically synthesized gRNA components with ATTO 550 dye, wherein fluorescently labeled tracrRNA can be used to detect and visualize cells that have been successfully transfected with an RNP (pg. 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 method for altering the genome of a plant comprising introducing into the plant cell an RNP complex taught by Bortesi to include a ATTO 550 dye fluorescent label on the tracrRNA , as taught by Schubert. One of ordinary skill in the art would have been motivated to add a fluorescent label on the tracrRNA taught by Bortesi to be able to detect and visualize plant cells that have been successfully transfected with an RNP with a higher delivery efficiency when compared to simply labeling the endonuclease. The rationale to support a conclusion that the claims would have been obvious is that all the claimed elements were known in the prior art, and one of ordinary skill could have combined these elements as claimed with no change to their respective functions. Thus the combination yielding predictable results would have been expected by a skilled artisan.
Regarding Claims 2 and 15, Bortesi teaches in two working examples (Example 1 and Example 2) that the plant cells that had the RNP complex delivered to them were observed using fluorescence microscopy to determine whether or not the delivery of the RNP complex to the cells resulted in efficient DNA cleavage (Example 1, paragraphs 0075-0077; Example 2, paragraphs 0078-0079).
Regarding Claim 3, Bortesi teaches in several working examples that plants cells which were identified to have undergone genome editing or modification due to the introduction of the RNP complex into said plant cell were subsequently regenerated into plants using various regeneration media (Example 5, paragraphs 0085-88; Example 8, paragraphs 0093-94; Example 9d, paragraphs 0100-101; Example 9F, paragraph 0103; Example 10b, paragraphs 0105-106).
Regarding Claim 8, Bortesi teaches that, regarding the method for altering the genome of an intact differentiated cell(s) without inserting exogenous genetic material into the genome, the nucleic acid modifying protein is a CRISPR-Cas9 endonuclease, a CRISPR-Cpf1 nuclease or a CRISPR-C2c2 endoribonuclease (pg. 24, Claim 13).
Regarding Claim 9, Bortesi teaches that, regarding the method for altering the genome of an intact differentiated cell(s) without inserting exogenous genetic material into the genome, the RNP-complex comprises a nucleic acid comprising a crRNA and a tracrRNA, or a chimeric cr/tracrRNA hybrid (pg. 24, Claim 14).
Regarding Claim 10, Bortesi teaches that, regarding the method for altering the genome of an intact differentiated cell(s) without inserting exogenous genetic material into the genome, the crRNA and tracrRNA, or the cr/tracrRNA hybrid, is targeted to a sequence that is endogenous to the cell; and a Cas9 endonuclease molecule that induces a double strand break at or near the sequence to which the crRNA and tracrRNA sequence is targeted, or at or near the sequence to which the cr/tracrRNA hybrid is targeted (pg. 24, Claim 14).
Regarding Claim 11, Bortesi teaches that, regarding the method for altering the genome of an intact differentiated cell(s) without inserting exogenous genetic material into the genome, the differentiated cell is exposed to the pre-assembled ribonucleoprotein (RNP)-complex, in particular in the presence of a carrier material, wherein after delivering said RNP-complex into the cell, the genome of the cell is altered/modified, and wherein after the modification/alteration the cells are further cultivated to derive a cell, cell line, tissue or organism with a modified (altered) genome without the presence of a marker gene (pg. 23, Claim 8).
Regarding Claim 13, Bortesi teaches that, regarding the method for altering the genome of an intact differentiated cell(s) without inserting exogenous genetic material into the genome, the genome-modifying formulation comprises a plurality of different pre-assembled ribonucleoprotein (RNP)-complex, in particular wherein the ribonucleoprotein (RNP)-complexes differ in the nucleic acid sequence of the ribonucleic acid comprised in the complexes (pg. 23, Claim 7).
Regarding Claims 14 and 18-19, in one embodiment of the invention, Bortesi teaches targeting two different regions of an endogenous tobacco gene (pds) using multiple preassembled RPNs, demonstrating that two target regions were efficiently and simultaneously modified (Example 4, paragraphs 0082 and 0084). Because Bortesi teaches targeting two different regions of an endogenous tobacco gene (pds), the regions would inherently have different nucleic acid sequences for the crRNA to bind to, and as such are targeted different “target sequences” in the genome of N. tabacum.
Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicant’s Remarks on pgs. 11-15 in the reply filed on 09/29/2025 are acknowledged but do not overcome these modified rejections. In particular, Applicant’s Remarks rely on the premise that although Bortesi proposes a laser-based approach for cell perforation, there is no description of how this would be performed. There is no description of where or how the laser would be focused, no description of timing or power, and no description of how transfection would occur. Applicant argues that the laser-based approach seems to be an unsupported wishful afterthought and without further detail, the artisan would have to conduct extensive and undue experimentation to arrive at Applicant's approach (Remarks, pg. 12, second paragraph).
This is not found to be persuasive as Applicant is reading limitations into the claims that do not
exist in their currently amended form. It is noted that Claims 1 and 4 (presumably the claims the Applicant is providing arguments for in this case), do not recite additional claim limitations that require detailing or defining where or how the laser would be focused, description of timing or power, or description of how transfection would occur. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Additionally, Bortesi explicitly recites that an embodiment of their invention includes physical delivery methods, such as perforation, wherein perforation of the RNP complex is performed with echo waves or a laser (paragraph 0011), however it is the combined teachings of Bortesi, Rukmana, and Schubert that render the claims prima facie obvious. Disclosed examples and preferred embodiments do not constitute a teaching away from a broader disclosure or nonpreferred embodiments. In re Susi, 440 F.2d 442, 169 USPQ 423 (CCPA 1971). "A known or obvious
composition does not become patentable simply because it has been described as somewhat inferior to
some other product for the same use." In re Gurley, 27 F.3d 551, 554, 31 USPQ2d 1130, 1132 (Fed. Cir.
1994). As such, even if the combined teachings of Bortesi, Rukmana, and Schubert is considered inferior, wherein the artisan would have to conduct extensive and undue experimentation to arrive at Applicant's approach, Bortesi still discloses that perforation of the plant cell to introduce and RNP complex can be performed with a laser (paragraph 0011).
Applicant also argues that the technical approach in Rukmana differs from those of the invention in that Rukmana pretreats the cells with enzymes, then porates the cells using a laser. In contrast, Applicant's approach does not require pretreating cells nor pore formation. Rather, Applicant's approach induces a transient permeabilization of the cell wall and membrane of intact plant cells, which is significantly more gentle than physical perforation (Remarks, pg. 13, paragraph 3).
This is not found persuasive. First, although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Applicant’s instant claims are not explicitly narrowed to embody the version of the invention that is described as “Applicant’s approach”. Second the comprising language of Claims 1 and 4 allows for there to be additional steps added to the method steps, and thus the enzyme pre-treatment of the cells and pore formation taught by Rukmana does not disqualify the disclosure of Rukmana or the obviousness rationale of the combination of Bortesi, Rukmana, and Schubert.
Applicant also argues that Rukmana's examples only injected green fluorescent nanoparticles, which are not functional complexes akin to Applicant's RNP-complex, which is a complex of functional protein and RNA. That is, a functional protein is not required in Rukmana because of the FITC label. As such, Rukmana would not induce one or more single or double stranded DNA breaks in the cell genome to produce a cell having a detectable targeted genomic modification as provided in claim 1 (Remarks, pg. 13, paragraph 4).
This is not found persuasive. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The disclosure of Rukmana is not individually relied on to teach the claim limitations that recite, in part, “providing a genome-modifying formulation comprising a pre-assembled ribonucleoprotein (RNP)-complex comprising a nucleic acid-modifying protein and ribonucleic acid, wherein the ribonucleic acid is labeled with a visual marker” or “inducing one or more single or double stranded DNA breaks in the cell genome to produce a cell having a detectable targeted genomic modification without the presence of any exogenous genetic material in the cell genome”. The claim limitations in the previous sentence are explicitly recited in the disclosure of Bortesi (See Bortesi; pg. 23, Claim 1), wherein Schubert provides evidence that practical, effective approach for RNP detection is to fluorescently label the chemically synthesized gRNA components with ATTO 550 dye, wherein fluorescently labeled tracrRNA can be used to detect and visualize cells that have been successfully transfected with an RNP (pg. 1). Additionally, Examiner would like to draw Applicant attention to the state of the prior art, which teaches that including florescent labels in RNP complexes delivered to plant cells (marker assisted selection) is well known and routine in the art (See He and Zhao; Abiotech 1.1 (2019): 88-96, pg. 92, paragraph bridging left and right column).
Applicant also argues that it would also require undue experimentation to arrive at selecting the cells transfected with the RNP-complex and regenerating the cells to intact plantlets by growing in and/or on a growth media. Applicant argues that Rukmana's approach is limited to cell lines, such as TBY-2 cells. It is well known that TBY-2 cells do not regenerate into intact plantlets (Remarks, pg. 13, paragraph 5).
This is not found persuasive. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The disclosure of Rukmana is not relied on to teach the claim limitations of Claim 3, comprising “selecting the cells transfected with the RNP-complex and regenerating the cells to intact plantlets by growing in and/or on a growth media”. Bortesi teaches in several working examples that plants cells which were identified to have undergone genome editing or modification due to the introduction of the RNP complex into said plant cell were subsequently regenerated into plants using various regeneration media (Example 5, paragraphs 0085-88; Example 8, paragraphs 0093-94; Example 9d, paragraphs 0100-101; Example 9F, paragraph 0103; Example 10b, paragraphs 0105-106). Therefore, Bortesi does teach cells lines that can be regenerated into plantlets. As such, it is the combined teachings of Bortesi, Rukmana, and Schubert that render Claim 3 prima facie obvious.
Regarding Claim 4, Applicant argues that one having ordinary skill in the art to which the invention belongs would not expect the success of Rukmana's free cell approach based on mannitol and enzymatic treatment for only ten minutes to equally apply to plant tissue and explants (Remarks, pg. 14, paragraph 3). Applicant also argue that Rukmana would also require the use of cells in culture rather than plant tissue or explants as provided in claim 4, and that Rukmana doesn’t teach tolerances of the laser-based approach that applies to tissue or explants (Remarks, pg. 14, paragraph bridging pgs. 14-15). Applicant also argues that the enzymatic treatment of Rukmana doesn’t readily transfer to plant tissue and explants due to the tight packing of cells, providing significantly less available cell surface area, and possible cuticle, and that such a treatment wouldn’t not logically work for plant tissue and explants, presumably one would at least be forced to significantly increase power, which Rukmana found deadly (Remarks, pgs. 14-15, paragraph bridging pgs. 14-15).
This is not found persuasive. First Applicant defines “explant” in the instant specification as including any portion of a plant including a plurality of intact plant cells derived by any [plant] organ and/or tissue of an organ (Specification, pg. 16, lines 29-30). As such, the Applicant’s instantly claimed “explant” recited in Claim 4 includes a plurality of plant cells, like those taught in Rukmana (plant cells derived from the seedlings of N. tabacum L. cv. Bright Yellow 2). Additionally, the arguments regarding Claim 4 are not commensurate to the scope of the currently amended claims. Within Claim 4, Applicant’s has not provided a requirement that every cell of the explant (or in the case of Rukmana, plurality of plant cells) that undergoes the laser-assisted transfection have an effective delivery of the RNP-complex, which is highlighted by the recitation Claim 4, part vi—meaning that even Applicant has some expectation that not all cells will be transfected with the RNP-complex if there needs to be a step to identify which ones have and have not. As such, even if the suspension of plant cells taught by Rukmana would be difficult to use in the method taught by Rukmana without a high level of optimization, that does not disqualify the disclosure of Rukmana which teaches successful optoporation of some number of plant cells after pre-treating the suspension of plant cells with enzymes. Additionally, the plurality of plant cells taught by Rukmana would not have the disadvantageous “tight packing of cells, providing significantly less available cell surface area, and possible cuticle” that Applicant argues explants would be expected to have.
Regarding Claim 6, Applicant argues that Applicant argues that there is no suggestion of how laser irradiation conditions for Applicant's system would be determined (Remarks, pg. 15, paragraph 2).
This is not found persuasive. Presumably the Applicant is urging that the disclosure of Rukmana— teaching that the laser irradiation conditions are adjusted prior to delivery of the biomolecule through the laser assisted transfection method (pg. 066104-2, NIR fs laser photoinjection)—does not provide a suggestion of how laser irradiation conditions for Applicant's instantly system would be determined. It appears that Applicant is reading limitations into the claims that do not exist in their currently amended form. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Instantly Claim 6 only requires that the “irradiation conditions are adjusted prior to delivering the RNP-complex by laser-assisted transfection. Instant Claim 6 does not require any particular laser irradiation conditions, merely that the conditions are adjusted prior to delivering the RNP-complex. As such, the disclosure of Rukmana comprises the instantly claimed limitation and renders Claim 6 prima facie obvious.
Claims 5 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Bortesi et al. (EP 3392339 A1, published 10/24/2018) in view of Schubert et al. ("Fluorescently labeled tracrRNA provides efficient genome editing while allowing cellular microscopy and FACS analysis" ©2017) and Rukmana et al. (APL photonics 5.6, published 06/15/2020) as applied to Claim 4 above, and further in view of Li et al. (Crop Breeding and Applied Biotechnology 18.02 (2018): 184-191) and Reddy et al. (Industrial Crops and Products 40 (2012): 324-335).
The teachings of Bortesi, Schubert, and Rukmana as they are applied to Claim 4 are set forth previously herein and are incorporated by reference.
However, Bortesi, Schubert, and Rukmana do not teach the feature of Claim 5, wherein the method of Claim 4 further comprises analyzing the plantlets to confirm editing of the target gene using any method for genetic characterization of genome-edited plants such as high-resolution melt analysis for identifications of mutated alleles; and selecting the analyzed plantlets scoring positive and further growing those plantlets to full plants.
Regarding Claims 5 and 16, Li et al. (herein referred to as Li) teaches a method of high resolution melting-facilitated rapid identification and genotyping of mutations induced by CRISPR/Cas9 mutagenesis in rice. Li teaches genetically modifying rice plant cells, wherein after genetic modification, plantlets were regenerated (single independent T0 plants), and for identification of transgene positive T0 plants, genomic DNA was extracted from leaf tissues using a modified CTAB method (pg. 185, last paragraph) and high resolution melt curve analysis was used to detect mutations in the target regions in the T0 plants (pg. 186, HRM identification of genetic variations). Seeds harvested from the T0 plant were grown into T1 populations and high resolution melt curve analysis was used to detect mutations in the target regions in the T1 plants (pg. 185, last paragraph; pg. 186, HRM identification of genetic variations).
Reddy et al. (herein referred to as Reddy) teaches that using HMR to identify genetic variants in tobacco is routine and known in the art (Abstract).
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 method for altering the genome of a plant comprising introducing into the plant cell an RNP complex taught by Bortesi to include additional steps to genetically characterize the plantlets that underwent genetic modification for evidence of genome editing using HMR before growing subsequent progeny populations of plants as taught by Li. Because of the combine teachings of Li and Reddy, one of ordinary skill in the art would have been motivated to add these additional steps because using HMR to identify genetic variants in tobacco is routine and known in the art (Reddy; Abstract) and HMR enables high-throughput screening of plantlets, making it more cost effective and taking less time when compared to the T7EI method or PAGE-based assays and SSCP (Li, pg. 190, second full paragraph). The rationale to support a conclusion that the claims would have been obvious is that all the claimed elements were known in the prior art, and one of ordinary skill could have combined these elements as claimed with no change to their respective functions. Thus the combination yielding predictable results would have been expected by a skilled artisan.
Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicant’s Remarks on pgs. 15-16 in the reply filed on 09/29/2025 are acknowledged but do not overcome these rejections for the reasons given above in regards to the 35 U.S.C. 103 rejection applied to Claims 1-4, 6-11, 13-14, and 18-19.
Claims 12 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Bortesi et al. (EP 3392339 A1, published 10/24/2018) in view of Schubert et al. ("Fluorescently labeled tracrRNA provides efficient genome editing while allowing cellular microscopy and FACS analysis" ©2017) and Rukmana et al. (APL photonics 5.6, published 06/15/2020) as applied to Claim 1 above, and further in view of Schinkel et al. (Biotechnology and bioengineering 99.1 (2008): 244-248).
The teachings of Bortesi, Schubert, and Rukmana as they are applied to Claim 1 are set forth previously herein and are incorporated by reference.
However, Bortesi, Schubert, and Rukmana do not teach the feature of Claim 12, wherein the laser is a multiphoton laser operating under pulsing conditions and wherein the laser power is between 0.5 W and 3 W, preferably about 2 W, more preferably wherein the laser power is between 65 and 90%, preferably at 70% of 2 W and wherein preferably the wavelength is between 700 nm und 900 nm, preferably 800 nm (See 35 U.S.C. 112(b) rejection above).
In reference to following rejection, it should be noted, according to https://www.gentec-eo.com/blog/laser-pulse-energy-calculation, that one watt (W) is equivalent to one joule per second (J/s) and that one hertz (Hz) is equivalent to one pulse per second.
Regarding Claim 12, Schinkel et al. (herein referred to as Schinkel) teaches laser-assisted methods for perforation of single Nicotiana tabacum plant cells, wherein the procedure was shown to be suitable for the efficient delivery of DNA expression constructs to the nucleus, as demonstrated by the subsequent expression and correct targeting of a recombinant fluorescent protein and results showing that isolated plant cells can be permeabilized without direct manipulation (pg. 244, Abstract). Schinkel teaches that the laser energy used in the methods of their invention is about 750 microjoules at 1000Hz (pg. 245, right column, first paragraph), which is 0.75 J/s or 0.75 watt (W)—which is within the range recited in the instant claim.
Regarding Claim 16, based on the teachings of Schinkel which are directed to laser-assisted transfection of plant cells, wherein the laser apparatus and energy can be adjusted for the parameters of a specific experiment, the prior art directs one of ordinary skill in the art to make any changes to the laser power and laser wavelength that leads to laser-assisted transfection of a plant cell. It is well within the experience of that skilled artisan to perform optimization or adjustment of the laser apparatus to have difference power and wavelength settings— ultimately to cause transfection of the plant cell. Examiner invites Applicant to submit evidence to show that the specific power settings and wavelength recited in Claim 17 does anything other than what is reasonably expected (i.e., allow transfection of a plant cell) based on the disclosure of the prior art and evidence of the instant specification.
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 laser power and laser irritation conditions that were adjusted for cell type and distance from the laser source as taught by Rukmana such that the laser power is from 0.5 to 3 J/s (0.5 W to 3 W) as taught by Schinkel or wherein the laser power is at 70% of 2 watts and the wavelength is 800 nm as rendered obvious by Schinkel. One of ordinary skill in the art would have been motivated to make such adjustments with a reasonable expectation of success because Schinkel achieved the efficient delivery of DNA expression constructs to the nucleus with such laser power.
Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicant’s Remarks on pgs. 11-15 in the reply filed on 09/29/2025 are acknowledged but do not overcome these modified rejections. In particular, Applicant’s Remarks rely on the premise that the settings in Schinkel would not apply to the invention as claimed. Applicant argues that Schinkel used a laser with energy lower than 5μJ as described in the material and methods "For the optoporation experiments, the beam was attenuated using a reflecting prism and a polarizing beam splitter to provide energy pulses of <5 μJ." (See pg. 245 "Optoporation Apparatus"). This would be <0,005 J/s at 1000 Hertz pulsing frequency which is equivalent to <5 mW i.e. a factor of 100 outside the range of the minimum laser power of the present invention. Applicant argues that Schinkel's best efficiency of optoporation was achieved at 600 nJ (See pg. 246 "Optoporation of Cells" "The laser beam was attenuated to 600 nj .... " and Results and Discussion on page 246 "The highest energy level that caused poration without damage of the protoplast was 600 nJ") which is 0.6 μJ equivalent to 0.6 mJoules/s with a 1000 Hertz pulsing frequency. This makes the power of the laser in the mWatt range i.e 0.6 mWatt or 600 μWatt which is largely out of the range in claim 14 (Examiner assumes Applicant means Claim 12), where the minimum power is 0.5 W (i.e 500 mW) to 3W (Remarks, pgs. 16-17).
This is not found persuasive. "[A] prior art reference that discloses a range encompassing a somewhat narrower claimed range is sufficient to establish a prima facie case of obviousness." In re Peterson, 315 F.3d 1325, 1330, 65 USPQ2d 1379, 1382-83 (Fed. Cir. 2003). As such, the Applicant’s own remarks acknowledge that an embodiment of the disclosure taught by Schinkel does use a multi-photon laser operating under pulsing conditions and wherein the laser power is 0.6 watts, which is within the range of laser powers (0.5 watts to 3 watts) recited in instant Claim 12. Without any experimental evidence, even if Applicant posits that “one of ordinary skill in the art would not achieve a successful transfection of intact plant cells based on the methods described by Schinkel”, "A known or obvious composition does not become patentable simply because it has been described as somewhat inferior to some other product for the same use." In re Gurley, 27 F.3d 551, 554, 31 USPQ2d 1130, 1132 (Fed. Cir.1994). Examiner invites Applicant to provide experimental evidence to show that the power settings taught by Schinkel would not lead to a successful transfection of a plant cell.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
---The following rejections are modified from those set forth in the Office Action dated 05/02/2025 in view of Applicant’s amendments to the claims dated 09/29/2025 and addition of new Claims 15-19---
Provisional Nonstatutory Double Patenting over App No. 18/026,102
Claims 1-6, 12-13, and 15 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over Claims 6-9 and 11-14 of co-pending Application No. 18/026,102 (US 2023/0357783, filed 03/13/2023). The co-pending and instant applications share multiple inventors and the same applicant.
Although the claims at issue are not identical, they are not patentably distinct from each other because:
Co-pending Claim 6 is drawn to method for introducing a biomolecule into an intact plant cell by a laser-assisted transfection method comprising the steps of: (i) providing a plant explant isolated by any organ and tissue of a plant and comprising intact plant cells or an isolated plant cell and selectable marker;(ii) providing a biomolecule and a selectable marker; and (iii) delivering said biomolecule and the selectable marker into the plant cell by a laser-assisted transfection method, wherein the cell or tissue is surrounded by a liquid and the laser focal point is in the liquid adjacent to the cell wall and not directly in contact with the cell wall. According to the co-pending specification (pg. 9, 1st para), the term "biomolecule" includes macromolecules (or polyanions) such as polypeptides, proteins, carbohydrates, lipids, and polynucleotides (nucleic acids) like DNA or RNA, as well as small molecules such as primary metabolites, secondary metabolites and natural products. According to the co-pending application, the term “selectable marker” includes reporter genes like fluorescent proteins (pg. 10, bottom of last paragraph).
Instant Claim 1 is drawn to method for altering the genome of an intact plant cell without inserting exogenous genetic material into the genome, the method comprising: (i) providing an intact plant cell with a cell wall, the plant cell comprising an endogenous gene to be modified; (ii) providing a genome-modifying formulation comprising a pre-assembled ribonucleoprotein (RNP)-complex comprising a nucleic acid-modifying protein and ribonucleic acid, wherein the ribonucleic acid is labeled with a visual marker; (iii) delivering said RNP-complex into the plant cell by a laser-assisted transfection method, wherein the cell is surrounded by a liquid and the laser focal point is in the liquid adjacent to the cell wall and not directly in contact with the cell wall; and (iv) inducing one or more single or double stranded DNA breaks in the cell genome to produce a cell having a detectable targeted genomic modification without the presence of any exogenous genetic material in the cell genome.
As such, the genome modifying formulation comprising an RNP-complex which comprises a nucleic acid-modifying protein and a ribonucleic acid of instantly pending Claim 1 encompasses two species from the genus of “biomolecules” as defined by the co-pending specification. Additionally the visual marker recited in instant Claim 1 encompasses a species from the genus of “selectable markers” as defined by the co-pending application. As such, co-pending Claim 6 recites a genus encompassing the species recited in the instant application, and anticipates instant Claim 1.
Instant Claim 4 is drawn to a for producing genome edited plants without inserting exogenous genetic material into the genome of the plant, the method comprising the steps of:(i) providing a plant explant isolated by any organ and tissue of a plant and comprising intact plant cells comprising an endogenous gene to be modified; (ii) providing a genome-modifying formulation comprising a pre-assembled ribonucleoprotein (RNP)-complex comprising a nucleic acid-modifying protein and ribonucleic acid, wherein the ribonucleic acid is labeled with a visual marker; (iii) layering the plant explant onto the RNP-complex formulation in a transparent support; (iv) delivering said RNP-complex into a plant cell of the plant explant by laser- assisted transfection, wherein the cell is surrounded by a liquid and the laser focal point is in the liquid adjacent to the cell wall and not directly in contact with the cell wall; (v) inducing one or more single or double stranded DNA breaks in the cell genome to produce a cell having a detectable targeted genomic modification without the presence of any exogenous genetic material in the cell genome; (vi) identifying the cells transfected with the RNP-complex by microscopy, in particular by fluorescence microscopy; (vii) selecting the cells transfected with the RNP-complex from the plant explant, and (viii) regenerating the cells to intact plantlets by growing in and/or on a growth media.
As such, the genome modifying formulation comprising an RNP-complex which comprises a nucleic acid-modifying protein and a ribonucleic acid of instantly pending Claim 4 encompasses two species from the genus of “biomolecules” as defined by the co-pending specification. Additionally the visual marker recited in instant Claim 4 encompasses a species from the genus of “selectable markers” as defined by the co-pending application. Additionally, the co-pending specification defines “intact plant cells” as comprising a cell wall, and can be obtained from cell aggregates or from plant tissue (pg. 14, second full paragraph). Therefore the “plant explant isolated by any organ and tissue of a plant and comprising intact plant cells” recited in instant Claim 4 is a species of the genus of “intact plant cells” recited in co-pending Claim 6. As such, co-pending Claim 6 recites multiple genera that encompass the species recited in the instant application, and anticipates instant Claim 4.
Co-pending Claim 7 anticipates the RNP-complex recited in instant Claims 1 and 4.
Co-pending Claim 8 anticipates the “identifying the cells” step of instant Claim 2 and the “analyzing the plantlets” step of instant Claim 5.
Co-pending Claim 9 anticipates the “visual marker” of instant Claims 1 and 4 and the “identifying the cells” step of instant Claims 2 and 4.
Co-pending Claim 11 anticipates the “selecting the cells” step of instant Claim 3.
Co-pending Claim 12 anticipates the “plurality of different pre-assembled RNP-complexes” recited in instant Claim 13.
Co-pending Claim 13 anticipates the “adjusted laser irradiation conditions” recited in instant Claim 6.
Co-pending Claim 14 anticipates the “laser power” recited in instant Claims 12 and 17.
Co-pending Claim 17 anticipates the “florescence microscopy” recited in instant Claim 15.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Provisional Nonstatutory Double Patenting over App No. 18/026,102 in view of Bortesi et al.
Claims 8-11 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over Claims 6-7, 11-12, and 15 of copending Application No. 18/026,102 (US 2023/0357783, filed 03/13/2023) in view of Bortesi et al. (EP 3392339 A1, published 10/24/2018).
The co-pending and instant applications share multiple inventors and the same applicant.
Co-pending Claim 6 is drawn to method for introducing a biomolecule into an intact plant cell by a laser-assisted transfection method comprising the steps of: (i) providing a plant explant isolated by any organ and tissue of a plant and comprising intact plant cells or an isolated plant cell and selectable marker; (ii) providing a biomolecule and a selectable marker; and (iii) delivering said biomolecule and the selectable marker into the plant cell by a laser-assisted transfection method, wherein the cell or tissue is surrounded by a liquid and the laser focal point is in the liquid adjacent to the cell wall and not directly in contact with the cell wall. According to the co-pending specification (pg. 9, 1st para), the term "biomolecule" includes macromolecules (or polyanions) such as polypeptides, proteins, carbohydrates, lipids, and polynucleotides (nucleic acids) like DNA or RNA, as well as small molecules such as primary metabolites, secondary metabolites and natural products. According to the co-pending application, the term “selectable marker” includes reporter genes like fluorescent proteins (pg. 10, bottom