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 .
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finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 05/13/2026 has been entered.
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 the Claims
Amendments dated 05/13/2026 have been entered.
Claim 7 is cancelled.
Claims 1-6 and 8-19 are pending.
Claims 1-6 and 8-19 are examined herein.
The provisional rejection of Claims 1-6 and 12-16 on the grounds of nonstatutory double patenting as being unpatentable over Claim 6-9 and 11-14 of co-pending Application No. 18/026,102 has been withdrawn in view of Applicant’s amendments to the claims dated 05/13/2026.
The provisional rejection of Claims 8-11 on the grounds of nonstatutory double patenting as being unpatentable over Claims 6-7, 11-12, and 15 of co-pending Application No. 18/026,102 in view of Bortesi et al. (EP 3392339 A1, published 10/24/2018) has been withdrawn in view of Applicant’s amendments to the claims dated 05/13/2026.
Claim Objections
Claims 1, 4, 10-12, 18, and 19 are objected to because of the following informalities:
Claim 1, part (iv), line 2 should be amended to recite “…to produce a plant cell having a detectable…”
Claim 4, part (v), line 2 should be amended to recite “…to produce a plant cell having a detectable…”
Claim 4, part (vi) should be amended to recite “…identifying the plant cells…”
Claim 4, part (vii), line 1 should be amended to recite “…selecting the plant cells…”
Claim 4, part (viii), line 1, should be amended to recite “…regenerating the plant cells into intact plantlets…”
Claim 10, line 3 should be amended to recite “…that is endogenous to the plant cell and that…”
Claim 11, line 1 should be amended to recite “…wherein the plant cell is…”
Claim 12, line 3 should be amended to recite “…wherein the wavelength is between 700 nm and 900 nm.”
Claim 18, line 2 should be amended to recite “…wherein the at least two different pre-assembled ribonucleoprotein (RNP)-complexes differ…”
Claim 19, line 1 should be amended to recite “…wherein the at least two different pre-assembled…”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
Indefiniteness
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.
---The following are new rejections from those set forth in the Office Action dated 12/18/2025, made in view of further examination of the claims. Applicant’s Remarks dated 05/13/2026 have been reviewed but are deemed inapposite to the new rejections.---
Claims 1-20 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. All dependent claims are included in these rejections unless the contain a limitation that overcome the deficiencies of the parent claim from which they depend.
Claims 1 and 4 recite the limitation wherein “the laser does not form a pore in the plant cell” which renders the claim indefinite. It is unclear to one of ordinary skill in the art if this limitation means the following: Is there a complete absence of pore formation in the plant cell (presumably, the plant cell wall and plant cell membrane) throughout the entirety of the laser assisted transfection method, temporary or otherwise? Or, does this limitation allow for “transient permeability” which would encompass temporary pore formation to introduce the RNP-complex into the plant cell, wherein the temporary pore is subsequently resealed through the plant cell’s natural wound repair process, ultimately rendering the plant cell having a detectable targeted genomic modification without the presence of any exogenous genetic material in the cell genome at the end of Claim 1 and Claim 4 part (v) without a pore. The instant specification does not define or recite the term “pore” (temporary or otherwise), and thus does not provide clarifying language for the metes and bounds of the invention (See 35 U.S.C. §112(a) rejection below). In fact, the only disclosure in the instant specification regarding how the RNP-complex is introduced into the claim plant cell recites, “laser-based method of the present disclosure it is not meant to induce abrasion or direct perforation of the wall and membrane of a plant cell, but rather a transient permeabilization of these two cell barriers” (pg. 5, lines 4-6). The specification does not define the term “transient permeabilization”; however, this is an art recognized term that is defined as the following:
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The art-recognized meaning of the term “transient permeabilization” creates a further lack of clarity to the claim limitation, as it seems the Applicant’s own specification requires at least temporary pore formation in the plant cell to achieve the delivery of the RNP-complex to the instantly claimed plant cells in Claims 1 and 4. For the reasons given above one of ordinary skill in the art would not be reasonably apprised on the metes and bounds of the claimed invention. For purposes of examination the instant claim limitation will be broadly interpreted to encompass temporary pore formation to introduce the RNP-complex into the plant cell, wherein the temporary pore is subsequently resealed through the plant cell’s natural wound repair process, ultimately rendering the plant cell having a detectable targeted genomic modification without the presence of any exogenous genetic material in the cell genome at the end of Claim 1 and Claim 4 part (v) without a pore. This determination does not relieve the applicant from their duty to amend the claims in any further correspondence.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, Claim 11 recites the broad recitation “modified”, and the claim also recites the term "altered" in parenthesis which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Claim 19 recites the limitation "...wherein the two different pre-assembled ribonucleoprotein (RNP)-complexes target different target sequences in the genome." in lines 1-3. There is insufficient antecedent basis for this limitation in the claim. Claim 15, from which Claim 19 depends, merely recites “The method according to claim 2, wherein the microscopy is fluorescence microscopy”, and does not explicitly reference any “different pre-assembled ribonucleoprotein(RNP)-complexes” as required by the dependency of Claim 19. For purposes of examination, Claim 19 will be interpreted as meaning to depend from instant Claim 14 rather than Claim 15. This determination does not relieve Applicant from their duty to amend the claim in any further correspondence.
Claim Rejections - 35 USC § 112
Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
---The following are new rejections from those set forth in the Office Action dated 12/18/2025, made in view Applicant’s amendments to the claims dated 05/13/2026. Applicant’s Remarks dated 05/13/2026 have been reviewed but are deemed inapposite to the new rejections.---
Claims 1-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. All dependent claims are included in these rejections unless the contain a limitation that overcome the deficiencies of the parent claim from which they depend.
Claims 1 and 4 have been amended to require the term “pore”.
While the laser-based method of the present disclosure describes ways an RNP-complex is not delivered into the instantly claimed plant cell (i.e., it is not meant to induce abrasion or direct perforation of the wall and membrane of a plant cell), as originally described in the specification, this particular type of plant cell perforation (unspecified “pore”), that is not supposed to occur in the methods of the invention, was not envisioned in the application as originally filed. There is no support in the specification for the amended claimed limitations. There is not even a single mention in the disclosure of the term “pore” , nor does there appear to be any implicit support that Applicant’s envisioned a plant cell that does not have this particular and unspecified type of cell wall and cell membrane perforation (temporary or otherwise). To this point, Applicant recites that the “laser-based method of the present disclosure it is not meant to induce abrasion or direct perforation of the wall and membrane of a plant cell, but rather a transient permeabilization of these two cell barriers” (pg. 5, lines 4-6). The specification does not define the term “transient permeabilization”; however, this is an art recognized term that is defined as the following:
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As such, the claim limitation wherein the laser does not form pores in the plant cells of Claims 1 and 4 directly opposes the method of the invention envisioned by Applicant in the instant specification. Simply, it appears a claim limitation that states “the laser does not form a pore in the plant cell” is the direct opposite of a laser that causes transient permeabilization of the cell wall and cell membrane.
Therefore the claims are rejected for introducing new matter in the application.
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 are new rejections from those set forth in the Office Action dated 12/18/2025, made in view Applicant’s amendments to the claims dated 05/13/2026. Applicant’s Remarks dated 05/13/2026 have been reviewed but are deemed inapposite to the new rejections.---
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, wherein the laser focal point is in the liquid adjacent to the cell wall and does not contact with the cell wall, wherein the laser does not form a pore in the plant cell, wherein passage of the RNP-complex across the plant cell wall and membrane is permitted only after laser irradiation of the liquid, 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). Rukmana also teaches that the pore created to allow the insertion of the target molecule into the plant cell is temporary and reseals itself (pg. 066104-6, left column, last sentence of first full paragraph). Also, Rukmana teaches that the laser photoinjection method used during experimentation increased the permeability of the plant cell wall (pg. 066104-2, left column, second full paragraph) and that the methods of the invention focus on the permeability of biological membranes (Abstract) as a whole.
In regard to the limitation of instant Claims 1 and 4, which states “wherein passage of the RNP-complex across the plant cell wall and membrane is permitted only after laser irradiation of the liquid”, the broadest reasonable interpretation of this limitation encompasses a method step wherein, only after the laser-assisted transfection can the biomolecule move through both the plant cell wall and membrane into the cell. Therefore, this claim limitation does not exclude steps taught by Rukmana prior to the laser photoporation, wherein the plant cell wall is altered (by mannitol and enzyme pre-treatment) prior to laser photoporation, because the teachings of Rukmana provide evidence that only after the use of the laser photoinjector did the target molecules actually diffuse through both the plant cell wall and plant cell membrane [FIG. 4(f) and FIG. 8(a)]— not through both the plant cell wall and plant cell membrane after the mannitol and enzymatic pre-treatment of the plant cells [FIG. 4(e) and FIG. 8(a)]. It is noted that the presence of fluorescence in the apoplast was seen in the pre-treated plant cells taught by Rukmana, after mannitol and enzymatic pre-treatment of the plant cells [FIG. 4(e)], however this result indicated that the permeability of the plant cell membrane did not allow diffusion of the target molecule through the plant cell membrane (pg. 066104-4, left column, last full paragraph) As such, the biomolecule diffusion through both the plant cell wall and membrane (“passage across the plant cell wall and membrane”) taught by Rukmana happens only after laser irradiation of the liquid, not before.
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). Based on the experimental results taught by Rukamana, the method laser photoporation taught by Rukmana would inherently cause the passage of the molecule of interest across both the plant cell wall and cell membrane only after the laser irradiation conditions wherein the laser focal point is focused on the culture medium directly adjacent to the cell wall and the pore formed by the laser irradiation reseals itself through natural plant cell repair mechanisms. 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 laser photoinjection, as taught by Rukmana.
However, the teachings of Bortesi and Rukmana do 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 RNPs, 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. 8-11 in the reply filed on 05/13/2026 are acknowledged but do not overcome these new rejections. In particular, Applicant’s Remarks rely on the premise that Bortesi proposes a laser-based approach for cell perforation, wherein 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 also urges that because Bortesi perforates the cell, Bortesi would not teach or suggest use of a laser that does not form a pore in the plant cell (Remarks, pg. 9).
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 do not recite additional claim limitations that require detailing or defining where or how the laser would be focused, or a description of timing or power. 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, 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). As shown in the new 35 U.S.C. 103 rejection above, the disclosure of Bortesi is not individually relied on to teach the claim limitation regarding the lack of pore formation in the plant cell or how transfection occurs (Also, see claim interpretation of this limitation in the 35 U.S.C. §112(b) rejection above). By modifying and/or optimizing the laser-based physical delivery methods taught by Bortesi, to have the laser focal point be focused on the culture medium directly adjacent to the cell wall, as taught by Rukmana, in the method of Rukmana that specifically focuses on the permeability of biological membranes (Abstract), one of ordinary skill in the art would reasonably expect that the temporary pore formed in the plant cell targeted by the laser irradiation method for delivery of a large target molecule would reseal itself through natural plant cell repair mechanisms. Examiner would also like to draw Applicant’s attention to the state of the prior art, which teaches that a diverse range of methods exist for permeabilizing the membrane of a cell for the insertion of foreign material, including using laser fields to open a transient pore in the membrane (photoporation). The prior art teaches that photoporation has successfully been demonstrated on a wide range of both animal and plant cell types, has numerous advantages, and that using a focused laser beam to create a sub-micron diameter self-healing pore in the plasma membrane of a cell for the selective introduction of membrane impermeable substances such as nucleic acids (e.g. DNA, mRNA, interference RNA) is well known and routine in the art (See US Patent No. 9,683,209 B2, issued 06/20/2017, column 1)
Applicant’s Remarks rely on the premise that the method taught by Rukmana does not teach or suggest "the laser does not contact the plant cell, further wherein the laser does not form a pore in the plant cell" (Remarks, pg. 9).
This is not found persuasive. In regards to the claim limitation wherein “the laser does not contact the plant cell”, the disclosure of Rukmana and Applicant’s explicit admission that Rukmana “teaches irradiation of TBY-2 cells at a distance of 10 pm from the cell wall in culture medium was successful but resulted in a low injection efficiency” provide evidence that Rukmana teaches the limitation wherein “ the laser does not contact the plant cell” [Remarks, pg. 9 and Rukmana pg. 066104-7, FIG. 8(a)]. ). "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 results of the laser photoinjection method taught by Rukmana are considered inferior to the instant method, Rukmana still discloses that the laser does not contact the plant cell [pg. 066104-7, FIG. 8(a)]. In regard to the claim limitation “wherein the laser does not form a pore in the plant cell”, Rukmana teaches that that the pore created to allow the insertion of the target molecule into the plant cell is temporary and reseals itself (pg. 066104-6, left column, last sentence of first full paragraph) (See 35 U.S.C. §112(b) rejection above for the claim interpretation of this limitation).
Applicant’s Remarks rely on the premise that the method taught by Rukmana does not teach or suggest "[passage] across the plant cell wall and membrane is permitted only after laser irradiation of the liquid" (Remarks, pg. 10).
This is not found persuasive. In regard to the limitation of instant Claims 1 and 4, which states “wherein passage of the RNP-complex across the plant cell wall and membrane is permitted only after laser irradiation of the liquid”, the broadest reasonable interpretation of this limitation encompasses a method step wherein only after the laser-assisted transfection can the biomolecule move through both the plant cell wall and membrane into the cell. Therefore, this claim limitation does not exclude steps taught by Rukmana prior to the laser photoporation, wherein the plant cell wall is altered (by mannitol and enzyme pre-treatment) prior to laser photoporation, because the teachings of Rukmana provide evidence that only after the use of the laser photoinjector did the target molecules actually diffuse through both the plant cell wall and membrane [FIG. 4(f) and FIG. 8(a)]— not through both the plant cell wall and membrane after the mannitol and enzymatic pre-treatment of the plant cells [FIG. 4(e) and FIG. 8(a)]. It is noted that the presence of fluorescence in the apoplast was seen in the pre-treated plant cells taught by Rukmana, after mannitol and enzymatic pre-treatment of the plant cells [FIG. 4(e)], however this result indicated that the permeability of the plant cell membrane did not allow diffusion of the target molecule through the plant cell membrane (pg. 066104-4, left column, last full paragraph) As such, the biomolecule diffusion through both the plant cell wall and membrane (“passage across the plant cell wall and membrane”) taught by Rukmana happens only after laser irradiation of the liquid, not before.
Applicant’s Remarks rely on the premise that Schubert does not use a laser-assisted approach where the laser focal point is directed to liquid adjacent to cells (Remarks, pg. 11).
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). As shown in the new 35 U.S.C. 103 rejection above, the disclosure of Schubert is not individually relied on the teach the claim limitation wherein there is as laser-assisted approach and wherein the laser focal point is directed to liquid adjacent to cells. It is the combination of Bortesi and Rukmana that teach those limitations, wherein Schubert is relied upon to teach that fluorescently labeled tracrRNA can be used to detect and visualize cells that have been successfully transfected with an RNP (pg. 1).
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 pg. 11 in the reply filed on 05/13/2026 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. Schinkel also explicitly states that the optoporation method used in the invention allowed for isolated plant cells to be permeabilized without direct manipulation (Abstract and pg. 247, right column, right full paragraph).
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, Schinkel explicitly states that the laser settings used in the method of their invention allowed for permeabilization of the isolated plant cells.
Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicant’s Remarks on pg. 11 in the reply filed on 05/13/2026 are acknowledged but do not overcome these modified rejections. In particular, Applicant’s Remarks rely on the premise that one of ordinary skill in the art would not apply Schinkel to the Applicant’s technical approach of permeabilization because Schinkel’s power setting is for cell perforation by irradiating the cells rather than plant cell permeabilization as claimed (Remarks, pg. 11).
This is not found persuasive, due to the fact that Schinkel explicitly teaches that the optoporation method used in the invention allowed for isolated plant cells to be permeabilized without direct manipulation (Abstract and pg. 247, right column, right full paragraph).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KELSEY L. MCWILLIAMS whose telephone number is (703)756-4704. The examiner can normally be reached M-F 08:00-17:30.
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/KELSEY L MCWILLIAMS/Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663
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