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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 01/30/2026 has been entered.
Election/Restrictions
The amendments received on 01/30/2026 have been entered. Claims 1, 4-6, 11-13, and 16-18 are pending.
Claims 11-12 and 17-18 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/20/2022.
Therefore claims 1, 4-6, 13 and 16 are examined in this Office Action.
For following analysis “substitute specification – Clean” received on 05/08/2023 has been used as reference when referring to page numbers and contents.
Following 35 USC § 103 rejection is modified to analyze the newly added limitation of the metal microparticles are “nonporous” and one or more microparticles are introduced into the maize plant via biolistic bombardment.
Claim Rejections - 35 USC § 112 – New Matter
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.
Claims 1, 4-6, 13 and 16 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 they include a limitation that overcomes the deficiencies of the parent claim.
Claim 1 recite the method would comprise introducing into the maize plant cell one or more nonporous metal microparticles.
Neither the instant specification nor the originally filed claims appear to provide support for the phrase/concept “nonporous metal microparticles”.
Specification have not described the phrase nonporous metal microparticles.
Specification provides support for a metal particle that are non-toxic, non-reactive, and that they have a lower diameter than the target cell (page20, second paragraph).
Hence there is not enough support for the method would comprise introducing into the maize plant cell one or more nonporous metal microparticles.
Thus, such a phrase/concept “nonporous metal microparticles” constitutes NEW MATTER. In response to this rejection, Applicant is required to point to support for the phrase/concept “nonporous metal microparticles” or to cancel the new matter.
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 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 non-obviousness.
Obvious over Svitashev et al., and further in view of Yang et al., Horlbeck et al., Tanaka et al., Bertrand et al., Brinker et al., Samuel et al., Anand et al.
Claims 1, 4-6, and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Svitashev et al., and further in view of Horlbeck et al. (Published Year: 2016, Journal: eLife, Volume: 5: e12677, pages: 1-21); and further in view of Yang et al. (Published Year: 2010, Journal: BMC plant biology, Vol. 10(1), pages: 1-11; and further in view of Tanaka et al. (Published Year: 2008, Journal: Plant physiology, 146(1), 149-161); and further in view of Bertrand et al. (US Publication No.: US 2014/0219925 A1, Publication date: Aug. 7, 2014), and further in view of Brinker et al. (US Publication No.: US 2018/0028686 A1, Publication date: Feb. 1, 2018), and Samuel et al. (US Patent No.: US 9,476,057 B2, Date of Patent: Oct. 25, 2016), and Anand et al. (US Publication No.: US 2017/0121722 A1, Publication date: May 4, 2017) and further in view of Joshi et al.(Published:2006, Journal: Langmuir 22, 300-305), and further in view of Klein et al. (Published: 1999, Journal: BIO/TECHNOLOGY 6: 559-563).
The claims are drawn to a method for genetic modification in a maize plant cell comprising (a) introducing into the maize plant cell one or more nonporous metal microparticles coated with a coating comprising: (i) a genome engineering component which comprises a double strand DNA break (DSB) inducing enzyme or a nucleic acid encoding same, or a single strand DNA break (SSB) inducing enzyme or a variant thereof selected from a CRISPR/Cas endonuclease, a CRISPR/Cpfl endonuclease, a zinc finger nuclease (ZFN), a homing endonuclease, a meganuclease and a TAL effector nuclease; and (ii) a second compound comprising one or more of: (ii.1) TSA , and (ii.2) a phytohormone 2,4-D, 6-BA and combination thereof, (iii) spermidine; and (b) cultivating the maize plant cell under condition allowing the genetic modification, wherein the genome engineering component (i) and second compound (ii) are transiently active and transiently present in the plant cell.
The claims are drawn wherein the method is performed without marker gene-based selection, and wherein the metal microparticle is introduced via biolistic bombardment.
In claim 1 recitation of term “microparticles” is interpreted as defined by applicant as “The term “particle bombardment” as used herein, also named "biolistic transfection" or "microparticle-mediated gene transfer" refers to a physical delivery method for transferring a coated microparticle or nanoparticle comprising a construct of interest into a target cell or tissue (Spec, page 26, last paragraph). Applicant further teaches the microparticle consists of a non-toxic, non-reactive material which can comprises metal such as gold or tungsten wherein the size of the microparticle may be in a range of 0.4-1.6 micron (μm), preferably 0.4-1.0 μm (Spec, page 30, paragraph 3).
Regarding claim 1, Svitashev et al. teaches a method for genetic modification in a maize embryo cells and regeneration of plants with the genetic modification (Svitashev et al., page 1, abstract). Svitashev et al. further teaches the method comprises a particle delivery to the maize embryo cells a genome editing (i.e. engineering) component a purified Cas 9 protein pre-assembled with in vitro transcribed gRNAs as ribonucleoprotein complex (gRNA and Cas9) (page 2, paragraph 3) and a second compound a DNA vector encoding cell division promoting transcription factors (maize ovule developmental protein 2 (OPD2) and a maize Wuschel (WUS) (Svitashev et al., page 5, right paragraph 4, Methods). OPD2 and WUS promote cell division hence they are proteins that cause improved plant regeneration from a somatic cell, a callus, or an embryonic cell. Svitashev et al. teaches their method is without DNA- and selectable markers produced regenerated plant with specifically targeted gene mutations and gene edits at high frequencies (Svitashev et al., page 2, right paragraph 2).
Since the method include ribonucleoprotein complex the introduction of the genome editing component is transient and it cannot be stably integrated into the plant genome. Furthermore, a To regenerants when crossed with wild-type Hi-II plants and the progeny were used for segregation analysis. Sexual transmission of mutated ms45 alleles at the expected Mendelian segregation (1:1) was demonstrated in all progeny plants analyzed which further showed the genome engineering component were transiently active or present (Svitashev et al., page 4, right paragraph 2).
Furthermore, applicant teaches “transient activity” is interpreted as defined by the applicant where "transient transformation" refers to the transfer of a foreign material [i.e., a nucleic acid fragment, protein, ribonucleoprotein (RNP), etc.] into host cells resulting in gene expression and/or activity without integration and stable inheritance of the foreign material. The genome engineering component is not permanently incorporated into the cellular genome, but provides a temporal action resulting in a modification of the genome (Substitute Spec-Clean, page 28, paragraph 3).
Svitashev et al. further teaches RNP delivery into maize cells and their cleavage were evaluated by harvesting embryos in 2 days after bombardment and total genomic DNA was extracted from these harvested embryos and fragments surrounding the targeted sequences were amplified by PCR and analyzed by amplicon deep sequencing and mutations were readily detected at all target sites when Cas9–gRNA RNP complexes were delivered (Svitashev, page 2, right paragraph 4, Table 1).
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Svitashev et al. teaches taking into consideration the high activity of RNP complexes in experiments, they attempted completely DNA-free genome editing without a selectable marker wherein Maize embryo cells were bombarded with Cas9–gRNA RNP complexes targeting three different genes: LIG, MS26 and MS45 and plants were regenerated and analyzed by sequencing for targeted mutations. Their results showed in all experiments mutant plants were recovered at surprisingly high frequencies ranging from 2.4% to 9.7% (Table 2), wherein no plants with off-site mutations were identified when Cas9 and gRNA were delivered as RNP complex (Table 2) (Svitashev et al., page 4, left paragraph 1-2 and right paragraph 1). Furthermore, primarily regenerated plants (T0) containing ms45 biallelic mutations were male sterile as expected given the requirement of MS45 for pollen development in maize22 (Fig. 3). T0 regenerants were crossed with wild-type Hi-II plants and the progeny were used for segregation analysis. Sexual transmission of mutated ms45 alleles at the expected Mendelian segregation (1:1) was demonstrated in all progeny plants analyzed (Svitashev et al., page 4, right paragraph 2). Hence Svitashev et al. teaches their method is performed without marker gene based selection.
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Furthermore, Svitashev et al. teaches maize regeneration from protoplasts remain unattainable or inefficient (Svitashev et al., page 2, left last paragraph) showing importance of regeneration in genetic modifications. Svitashev et al. teaches RNP complexes of Cas9 and gRNA can be delivered in maize embryo cells by particle bombardment which resulted in specifically targeted gene mutations and gene edits at high frequencies (Svitashev et al., page 2, left paragraph 2). For example, when the biolistic delivery of Cas9-gRNA RNP in maize cells was used to target four different genes including, liguleless1 (LIG), acetolactate synthase (ALS2), and male fertility genes MS26 and MS45. An editing efficiency comparable to DNA plasmids, ranging from 0.21 to 0.69% was observed for all four target sites in maize immature embryo cells which was comparably higher than for genes of MS45 and LIG and were comparable for other remaining genes (see Table 1 above). Hence someone skilled in the art would use the particle delivery method for delivering second compounds.
Svitashev et al. teaches the gold (i.e. metal) microparticles (0.6 µm) coated with RNA RNP complex, helper genes, cell division promoting transcription factors were introduced into the maize embryo cells via biolistic bombardment using helium gene gun (page 2, right paragraphs 2-3, page 5, right last paragraph).
Svitashev et al. does not teach a second compound can be a TSA and a phytohormone selected from 2, 4-D, 6-benzylaminopurine, zeatin and combinations.
Horlbeck et al. teaches highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy (page1, abstract). By adding or exposing a chromatin remodeling enzyme yeast chromodomain helicase DNA binding 1 (yChd1) to nucleosome Horlbeck et al. restored the Ca9 access to DNA in vitro and in vivo (page 12, paragraph 3). Horlbeck et al. further teaches their results contribute a first step in understanding and exploiting how chromatin affects Cas9 activity to enable more sophisticated and precise rules for targeting Cas9 (page 12, paragraph 4).
In maize treatment with TSA an increase in global histone acetylation and decrease in global DNA has been found. Yang et al. teaches treatment with trichostatin A, which inhibits histone deacetylases resulted in increased histone H4 acetylation accompanied by the recondensation of interphase chromatin and a decrease in both global H3K9 demethylation and DNA methylation during mitosis in maize root tip cells (page 1, Abstract). Hence someone skilled in the art would utilize TSA which has been found effective to remodel chromatin in maize that would increase access to gRNA, Cas9 activity and improve overall efficiency of genome editing as taught by Horlbeck.
Furthermore, Tanaka et al. teaches treatment of seeds with TSA resulted in the arrest of post germination growth and the growth-arrested seeds began to form true leaves with embryo-like structures on their surfaces in Arabidopsis (Fig. 3, A and B, see figure below), where as a strong expression of all the embryo related genes LEC1, LEC2, FUS2 etc. were detected in the embryo-like structures (Fig 3C) (page 151, right paragraph 1 and 2).
Horlbeck et al., Yang et al., and Tanaka et al. do not teach TSA is coated in a microparticles.
Furthermore, it would be obvious to coat TSA in microparticles and introduce into the maize plant cell. Bertrand et al. teaches particles of polymer chain P each comprising the molecular formula (I) (claim 1), which are spherical having an average diameter in the range from about 5nm to about 100 µm (page 4, paragraph 0085), 50 to 500 (i.e., 0.5 micron), 300 nm (i.e., 0.3 micron) (page 4, paragraph 0086). Bertrand et al. claim 1 teaches nanovector comprises a active ingredient which is an epigenetic modulator wherein claim 7 teaches the epigenetic modulator is trichostatin A (TSA). Bertrand et al. teaches one aspect of the invention is to supply nanovectors, in the form of polymer comprising at least one active ingredient, in particular an epigenetic modulator which is capable of penetrating into a cell (page 2, paragraph 0018).
Bertrand et al. teaches Once the active ingredient has been released the polymer chain P and the constituents other than the active ingredient or the detecting probe can be eliminated from the cell (page 7, paragraph 0135). Hence it would be transiently active it the cell.
Furthermore, Brinker et al. teaches a Mesoporous silica nanoparticles (MSN) comprising a metal oxide core and a CRIPSR component as cargo (claim 1), a guide component, a nuclease (claim 6) and further comprising a trichostatin A (TSA) (claims 12 and 15). Brinker et al. teaches packaging DNA with gold nanoparticles for example NanoFlares- an engineered DNA and metal complex in which the core the core of the nanoparticle is gold (page20, paragraph 0150). Brinker et al. teaches a complex comprising a nanoparticles e.g. Gold or metal nanoparticles that is used to package the DNA (page 26, paragraph 0208).
Horlbeck et al., Yang et al., Tanaka et al. and Bertrand et al. and Brinker et al. teaches coating microparticle with Spermidine.
Furthermore, Klein et al. teaches spermidine that was used to precipitate or adsorption of DNA to the surface of the microprojectiles and used for condensing materials that would be less susceptible to degradation by intracellular nucleases, influenced the efficiency of DNA delivery and increased expression units ( page 560, right paragraph3 and page 561 paragraphs 1-2). Klein et al. teaches using 1.2 µm microprojectile (page 559, right last paragraph). Klein et al. teaches the suspension cells were bombarded with tungsten particles coated with DNA and Spermidine (page563, left paragraph 2). Therefore, the coating comprising Spermidine would have been the routine laboratory molecule for efficient delivery of the DNA using microparticles.
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Brinker et al. teaches their cargo also comprise a hormone (paragraph 0229). Brinker et al. teaches the CRISPR/Cas system has been used in plants (page 32, paragraph 0322). Brinker et al. teaches the nanoparticulate has size ranges between 1-500 nm (i.e. 0.5 µm) (page 9, paragraph 0072). Brinker et al. teaches plant hormone, plays crucial roles in various aspects of the regulation of plant growth and development (page 1616, left last paragraph). Brinker et al. teaches MSNs are efficient tool for delivering chemicals into plants in a controllable way (page1625, left paragraph 1).
Furthermore, Joshi et al. teaches gold nanoparticles are successfully used for transmucosal insulin hormone delivery (page 301, Abstract, Scheme 1). Joshi et al. teaches nanogold- insulin would permeate in mucosal cells (page 304, left last paragraph). Joshi et al. teaches Nanoparticles could also serve as excellent delivery vehicles for a variety of biomolecules such as proteins, DNA, and drugs (page 300, left paragraph 1). Joshi et al. teaches tunable shape- and size-dependent optical properties of gold nanoparticles have been exploited in various surface coatings and biomedical applications which are biocompaticle, nontoxic, bind readily to a large range of biomolecules such as amino acids, proteins/enzymes, and DNA, and expose large surface areas for the immobilization of such biomolecules (page 300, left paragraph 2). Joshi et al. teaches the ability to modulate the surface chemistry of gold nanoparticles by binding suitable ligands has important applications in many areas such as novel organic reactions, sensors (both inorganic and biological entities), drug/DNA delivery, and imaging (page 300, right paragraph1). Therefore it would have been obvious to use metal microparticles such as nonporous gold microparticles to deliver CRISPR components, phytohormones and TSA.
Furthermore, Samuel et al. teaches introducing in a plant cell having cell wall, gold nanoparticles coated with molecule of interest (i.e. hormones) (claims 1 and 9). Samuel et al. teaches the molecule of interest can be any hormones (Col.5, lines 51-58),
Anand et al. teaches phytohormones auxin as 2,4-dichlorophenoxyacetic acid (2,4-D), cytokinins as e benzylaminopurine (BAP) and zeatin are effective phytohormones to produce regenerable plant structures that form directly from singe cell (page18, paragraph 0052, page 44-45, table 9).
Thus it would have been obvious to one of ordinary skill of art before the effective filing date of the claimed invention to simply substitute in Svitashev et al.’s method of genetic modification in a maize plant cell comprising introducing into the maize plant cell one or more metal microparticles coated with a coating comprising: (i) a genome engineering component as a RNP complex and another component as the cell division promoting transcription factors (OPD2 and WUS) taught by Svitashev et al. with Spermidine as taught by Klein et al. TSA as taught by Horlbeck et al., Yang et al., Tanaka et al., Bartrand et al., Brinker et al.., Anand et al., Klein et al. and Joshi et al. with predictable result of increased genome engineering efficiency and improved somatic regeneration, wherein the RNP complex would be transiently active and transiently present and the method would be selectable marker free method as taught by Svitashev et al.
Furthermore, the treatment with the mass of 15 ng to 45 ng Trichostatin A (TSA), and 125 ng to 500 ng of a phytohormone selected from 2,4-Dichlorophenoxyacetic acid, 6-benzylaminopurine, zeatin, and combinations thereof, would have been obvious to try for microparticles with the Applicant disclosed size of the microparticle in a range of 0.4-1.6 micron (μm), preferably 0.4-1.0 μm (Spec, page 30, paragraph 3), such small microparticle also showed by Klein et al. with microparticle diameter size of 0.6, 1.2 μm (page 561, right paragraph 2). It would have been obvious from the finite number of identified, predictable solutions, with a reasonable expectation of finding the specific mass range of TSA and the phytohormones for coating using the microparticles in the method of genetic modification in a maize plant cell.
Furthermore, the method would include cultivating the maize plant cell under conditions allowing the genetic modification of the genome of said maize plant cell by the transiently active genome engineering component as taught by Svitashev et al. would have resulted in the recited method for genetic modification in a maize plant cell. Furthermore, since TSA does not stably integrate in the genome it is a transient introduction.
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Regarding claim 4, Svitashev et al. teaches a method where the modification of the genome is selected from the most prevalent mutation types a deletion of one or two nucleotide, and an insertion in LIG gene or their combination while using a transient marker free method using RNPs (page 3, Figure 1).
Regarding claim 5, Svitashev et al. teaches DSBs in eukaryotic cells are repaired using two major pathways: non-homologous end joining (NHEJ) and homology-directed repair. Svitashev et al. further teaches about the method of differentiating mutations in their experiment for classifying as NHEJ mutation (page 6, paragraph 3).
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Regarding claim 6, Svitashev et al. teaches a method where the modification of the genome is selected from the most prevalent mutation types a deletion of one or two nucleotide, and an insertion in LIG gene or their combination (page 3, Figure 1).
Regarding claim 13, Svitashev et al. teaches a method for producing a genetically modified maize plant, comprising the steps of genetically modifying a maize plant cell by co-introducing RNP and maize ovule development protein and maize Wuschel protein (page 5, right paragraph 4, Methods) and regenerating a plant from the modified cell (page 5, right paragraph 7). Furthermore, Svitashev et al. teaches the genetic analysis of their regenerated T0 generation with expected mendelian ratio of 1:1 further showed the activity of genome engineering component were transient (page 4, right paragraph 2).
The method of producing genetically modified plant by modifying a maize plant cell, regenerating maize plant that does not contain any of the genome engineering component and second compound for example TSA used for co-introduction in the cell would not stably transfer to next generation and application of cointroduction of genome engineering component with TSA would be obvious over the teaching from Svitashev et al., Yang et al., Tanaka et al. and motivation from Horlbeck et al.
Obvious over Svitashev, and further in view of Kareem et al., Prasad et al., and, Tsuwamoto et al., Horlbeck et al., Tanaka et al., Yang et al., Bertrand et al., Brinker et al., Samuel et al., Joshi et al., Klein et al. and Anand et al.
Claims 1 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Svitashev et al. (Published Year: 2016, Journal: Nature communications, Volume: 7(1), pages: 1-7), and further in view of Tsuwamoto et al.(Published: 2010, Journal: Plant molecular biology, Vol. 73, pages: 481-492), and further in view of Prasad et al. (Published Year: 2011, Journal: Current Biology, Volume: 21, pages: 1123-1128), and further in view of Horlbeck et al. and further in view of Yang et al.; and further in view of Tanaka et al. and further in view of Bertrand et al. and further in view of Brinker et al., and further in view of Anand et al., and further in view of Joshi et al., and further in view of Samuel et al., and further in view of Klein et al.
The claims are drawn to a method for genetic modification in a maize plant cell comprising (a) introducing into the maize plant cell one or more nonporous metal microparticles coated with a coating comprising: (i) a genome engineering component which comprises a double strand DNA break (DSB) inducing enzyme or a nucleic acid encoding same, or a single strand DNA break (SSB) inducing enzyme or a variant thereof selected from a CRISPR/Cas endonuclease, a CRISPR/Cpfl endonuclease, a zinc finger nuclease (ZFN), a homing endonuclease, a meganuclease and a TAL effector nuclease; and (ii) a second compound comprising one or more of: (ii.1) TSA , (ii.2) a phytohormone 2,4-D, 6-BA and combination thereof, (iii) spermidine, and (b) cultivating the maize plant cell under condition allowing the genetic modification, wherein the genome engineering component (i) and second compound (ii) are transiently active and transiently present in the plant cell.
The claims are further drawn to the method wherein the method further comprises a protein causing improved maize plant regeneration from a somatic cell, a maize callus cell or maize embryogenic cell or an expression cassette comprising a nucleic acid encoding said protein, where in the protein causing improved maize plant regeneration comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1 encoded by nucleic acid sequence with at least 95% sequence identity to SEQ ID NO: 2.
Following analysis is based on the method for genetic modification in maize plant cell which would require introducing into the maize plant cell one or more metal microparticles coated with a coating comprising: genome engineering component (claim 1 item i) and a second compound comprising TSA and furthermore the second compound comprises an expression cassette comprising a nucleic acid encoding a protein causing improved maize plant regeneration from a maize cell.
In claim 1 recitation of term “microparticles” is interpreted as defined by applicant as “The term “particle bombardment” as used herein, also named "biolistic transfection" or "microparticle-mediated gene transfer" refers to a physical delivery method for transferring a coated microparticle or nanoparticle comprising a construct of interest into a target cell or tissue (Spec, page 26, last paragraph). Applicant further teaches the microparticle consists of a non-toxic, non-reactive material which can comprises metal such as gold or tungsten wherein the size of the microparticle may be in a range of 0.4-1.6 micron (μm), preferably 0.4-1.0 μm (Spec, page 30, paragraph 3).
Regarding claims 1 and 16, see analysis above for obvious method of genetic modification in a maize plant cell comprising introducing into the maize plant cell comprising microparticles coated with a coating comprising a genome engineering component and a TSA obvious over Svitashev et al., and further in view of Horlkbeck et al., Tanaka et al., Yang et al., Bertrand et al., Brinker et al., Klein et al., Joshi et al. and Anand et al.
Horlkbeck et al., Tanaka et al., Yang et al., Bertrand et al., Brinker et al., Samuel et al., Klein et al., Joshi et al., and Anand et al. do not teach the second compound further comprises an expression cassette comprising a nucleic acid encoding a protein causing improved maize plant regeneration from a maize somatic cell is an amino acid sequence of SEQ ID NO: 1 encoded by nucleic acid sequence of SEQ ID NO: 2 wherein it is transiently present or transiently active.
Tsuwamoto et al. teaches an Arabidopsis EMBRYOMAKER (EMK) (or AtEMK) gene which is homologous to one of the identified Brassica ESTs (BnGemb-58) and encodes an AP2 domain transcription factor (Page 481, Abstract) and it is identical to ANTIGUMENTA-LIKE 5 (AIL5) (page 485, right paragraph 3).
Tsuwamoto et al. teaches transient expression of AtEMK fused to sGFP as construct (page 483, right paragraph 2) where the transient and stable introgression was carried out with Biolistic PDS=1000/He a particle delivery system (page 483, right last paragraph) in Arabidopsis thaliana ecotype Columbia (Col-0) (page 482, right paragraph 3). Tsuwamoto teaches gold particles 1.6 µm in diameter were coated with the vectors and released to target (page 483, right last paragraph).
Tsuwamoto et al. teaches in emk mutant ectopic expression of AtMEK induced embryo-like structures from cotyledons and in invitro culture, AtMEK enhanced the efficiency of somatic embryogenesis (page 481, Abstract) and it is a factor promoting embryogenesis and may act in various embryogenesis (page 489, right paragraph 1, see Figure 6 below) wherein the AtMEK overexpressed like AtEMKox-2 showed higher percentage of callus with somatic embryo as compared to wild type and emk mutant lines (see Figure 6 below).
Tsuwamoto et al. teaches AtMEK was harmful for the normal development of plant after germination (page 481, Abstract) requiring transient transformation.
Tsuwamoto et al.’s AtEMK is the SEQ ID NO: 13 taught by applicant as boost gene as PLT5 in Arabidopsis which are taught as SEQ ID NOs: 1, 2, 13 and 14 (Spec, page 23, last paragraph, see figure below).
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Prasad et al. teaches the data indicate that PLT5 can be regulated by local auxin addition in a PIN1-independent mechanism (page 1127, left paragraph 2). Prasad et al. teaches their PLT5 gene nomenclature has ANTEGUMENTA-like (AIL) genes with name of AIL5 with ATG number of ATG57390 (Prasad et al., Supplementary Table S1). Where the ATG57390, see row DR below for TAIR database, has 100% sequence identity with SEQ ID NO: 13 an applicant described booster gene (see alignment below). Prasad et al. furthermore teaches PLT genes from monocots forms separate subclades which included additional amino acid positions where cluster including PLT5 (also known as AIL5) encompasses both monocot for example maize (Zea mays) PLT5 (Prasad, page 1123, left paragraph 1, Supplementary Figure S1). Furthermore, SEQ ID NO: 1 has 100% sequence identity to maize PLT5 (AIL5) as AP2-like ethylene-responsive transcription factor AIL5 (see alignment below).
Alignment to SEQ ID NO: 13 to UniProt 202202 database:
RESULT 1
AIL5_ARATH
ID AIL5_ARATH Reviewed; 558 AA.
AC Q6PQQ3; Q9FIE2;
DT 12-JUN-2007, integrated into UniProtKB/Swiss-Prot.
DT 12-JUN-2007, sequence version 2.
DT 25-MAY-2022, entry version 113.
DE RecName: Full=AP2-like ethylene-responsive transcription factor AIL5;
DE AltName: Full=Protein AINTEGUMENTA-LIKE 5;
GN Name=AIL5; OrderedLocusNames=At5g57390; ORFNames=MSF19.5;
OS Arabidopsis thaliana (Mouse-ear cress).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Brassicaceae; Camelineae; Arabidopsis.
OX NCBI_TaxID=3702;
RN [1]
RP NUCLEOTIDE SEQUENCE [MRNA].
RA Pan Y., Gong W., Liu D., Fu Q., Mei W.-Q., Song W.-Q., Ma L.-G., Luo J.-C.,
RA Deng X.-W., Zhu Y.-X.;
RT "Molecular cloning, expression, phylogenetic and functional
RT characterization of the Arabidopsis AP2/EREBP transcription factor
RT family.";
RL Submitted (MAR-2004) to the EMBL/GenBank/DDBJ databases.
RN [2]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Columbia;
RX PubMed=10048488; DOI=10.1093/dnares/5.6.379;
RA Asamizu E., Sato S., Kaneko T., Nakamura Y., Kotani H., Miyajima N.,
RA Tabata S.;
RT "Structural analysis of Arabidopsis thaliana chromosome 5. VIII. Sequence
RT features of the regions of 1,081,958 bp covered by seventeen physically
RT assigned P1 and TAC clones.";
RL DNA Res. 5:379-391(1998).
RN [3]
RP GENOME REANNOTATION.
RC STRAIN=cv. Columbia;
RX PubMed=27862469; DOI=10.1111/tpj.13415;
RA Cheng C.Y., Krishnakumar V., Chan A.P., Thibaud-Nissen F., Schobel S.,
RA Town C.D.;
RT "Araport11: a complete reannotation of the Arabidopsis thaliana reference
RT genome.";
RL Plant J. 89:789-804(2017).
RN [4]
RP FUNCTION, TISSUE SPECIFICITY, AND DEVELOPMENTAL STAGE.
RX PubMed=15988559; DOI=10.1007/s11103-005-0955-6;
RA Nole-Wilson S., Tranby T.L., Krizek B.A.;
RT "AINTEGUMENTA-like (AIL) genes are expressed in young tissues and may
RT specify meristematic or division-competent states.";
RL Plant Mol. Biol. 57:613-628(2005).
RN [5]
RP GENE FAMILY, AND NOMENCLATURE.
RX PubMed=16407444; DOI=10.1104/pp.105.073783;
RA Nakano T., Suzuki K., Fujimura T., Shinshi H.;
RT "Genome-wide analysis of the ERF gene family in Arabidopsis and rice.";
RL Plant Physiol. 140:411-432(2006).
CC -!- FUNCTION: Probably acts as a transcriptional activator. Binds to the
CC GCC-box pathogenesis-related promoter element. May be involved in the
CC regulation of gene expression by stress factors and by components of
CC stress signal transduction pathways (By similarity). Involved in the
CC regulation of floral organs size. {ECO:0000250,
CC ECO:0000269|PubMed:15988559}.
CC -!- SUBCELLULAR LOCATION: Nucleus {ECO:0000305}.
CC -!- TISSUE SPECIFICITY: Expressed in roots, seedlings, inflorescence, and
CC siliques. Also detected at low levels in leaves.
CC {ECO:0000269|PubMed:15988559}.
CC -!- DEVELOPMENTAL STAGE: Present in inflorescence meristem and later in
CC young floral mersitems. Expressed in sepal, petal, stamen and carpel
CC primordia. In petal, progressively confined to petal margin and
CC epidermal cells. Restricted to sporogenous tissue in the stamen and to
CC the medial ridge of the carpel. Present in tissues that develop from
CC this ridge, such as placenta and ovule primordia. In ovules, first
CC expressed in distal part of the funiculus and the outer integument,
CC before being confined to the funiculus. {ECO:0000269|PubMed:15988559}.
CC -!- SIMILARITY: Belongs to the AP2/ERF transcription factor family. AP2
CC subfamily. {ECO:0000305}.
CC -!- SEQUENCE CAUTION:
CC Sequence=BAB08476.1; Type=Erroneous gene model prediction; Evidence={ECO:0000305};
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DR EMBL; AY585685; AAS97942.1; -; mRNA.
DR EMBL; AB016891; BAB08476.1; ALT_SEQ; Genomic_DNA.
DR EMBL; CP002688; AED96895.1; -; Genomic_DNA.
DR RefSeq; NP_200549.2; NM_125122.4.
DR AlphaFoldDB; Q6PQQ3; -.
DR BioGRID; 21089; 1.
DR IntAct; Q6PQQ3; 1.
DR STRING; 3702.AT5G57390.1; -.
DR PaxDb; Q6PQQ3; -.
DR ProteomicsDB; 244935; -.
DR EnsemblPlants; AT5G57390.1; AT5G57390.1; AT5G57390.
DR GeneID; 835845; -.
DR Gramene; AT5G57390.1; AT5G57390.1; AT5G57390.
DR KEGG; ath:AT5G57390; -.
DR Araport; AT5G57390; -.
DR TAIR; locus:2173009; AT5G57390.
DR eggNOG; ENOG502QR5R; Eukaryota.
DR HOGENOM; CLU_013549_5_0_1; -.
DR InParanoid; Q6PQQ3; -.
DR OMA; DYWSNIF; -.
DR OrthoDB; 455685at2759; -.
DR PhylomeDB; Q6PQQ3; -.
DR PRO; PR:Q6PQQ3; -.
DR Proteomes; UP000006548; Chromosome 5.
DR ExpressionAtlas; Q6PQQ3; baseline and differential.
DR Genevisible; Q6PQQ3; AT.
DR GO; GO:0005634; C:nucleus; IDA:TAIR.
DR GO; GO:0003700; F:DNA-binding transcription factor activity; ISS:TAIR.
DR GO; GO:0000976; F:transcription cis-regulatory region binding; IPI:TAIR.
DR GO; GO:0060774; P:auxin mediated signaling pathway involved in phyllotactic patterning; IGI:TAIR.
DR GO; GO:1990110; P:callus formation; IGI:TAIR.
DR GO; GO:0009873; P:ethylene-activated signaling pathway; IEA:UniProtKB-KW.
DR GO; GO:0010311; P:lateral root formation; IGI:TAIR.
DR GO; GO:0060772; P:leaf phyllotactic patterning; IGI:TAIR.
DR GO; GO:0060771; P:phyllotactic patterning; IGI:TAIR.
DR GO; GO:0040019; P:positive regulation of embryonic development; IMP:TAIR.
DR GO; GO:0009791; P:post-embryonic development; IMP:TAIR.
DR GO; GO:0048364; P:root development; IMP:TAIR.
DR GO; GO:0009845; P:seed germination; IMP:TAIR.
DR CDD; cd00018; AP2; 2.
DR Gene3D; 3.30.730.10; -; 2.
DR InterPro; IPR001471; AP2/ERF_dom.
DR InterPro; IPR036955; AP2/ERF_dom_sf.
DR InterPro; IPR016177; DNA-bd_dom_sf.
DR Pfam; PF00847; AP2; 2.
DR PRINTS; PR00367; ETHRSPELEMNT.
DR SMART; SM00380; AP2; 2.
DR SUPFAM; SSF54171; SSF54171; 2.
DR PROSITE; PS51032; AP2_ERF; 2.
PE 2: Evidence at transcript level;
KW Activator; DNA-binding; Ethylene signaling pathway; Nucleus;
KW Reference proteome; Repeat; Transcription; Transcription regulation.
FT CHAIN 1..558
FT /note="AP2-like ethylene-responsive transcription factor
FT AIL5"
FT /id="PRO_0000290365"
FT DNA_BIND 203..269
FT /note="AP2/ERF 1"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00366"
FT DNA_BIND 305..363
FT /note="AP2/ERF 2"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00366"
FT REGION 1..65
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT REGION 74..93
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT REGION 166..195
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT REGION 387..406
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT COMPBIAS 1..56
FT /note="Polar residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT COMPBIAS 74..89
FT /note="Polar residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT CONFLICT 13
FT /note="Missing (in Ref. 1; AAS97942)"
FT /evidence="ECO:0000305"
SQ SEQUENCE 558 AA; 60306 MW; 3E215E06291CBD31 CRC64;
Query Match 100.0%; Score 2952; DB 1; Length 558;
Best Local Similarity 100.0%;
Matches 558; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MKNNNNKSSSSSSYDSSLSPSSSSSSHQNWLSFSLSNNNNNFNSSSNPNLTSSTSDHHHP 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MKNNNNKSSSSSSYDSSLSPSSSSSSHQNWLSFSLSNNNNNFNSSSNPNLTSSTSDHHHP 60
Qy 61 HPSHLSLFQAFSTSPVERQDGSPGVSPSDATAVLSVYPGGPKLENFLGGGASTTTTRPMQ 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 HPSHLSLFQAFSTSPVERQDGSPGVSPSDATAVLSVYPGGPKLENFLGGGASTTTTRPMQ 120
Qy 121 QVQSLGGVVFSSDLQPPLHPPSAAEIYDSELKSIAASFLGNYSGGHSSEVSSVHKQQPNP 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 QVQSLGGVVFSSDLQPPLHPPSAAEIYDSELKSIAASFLGNYSGGHSSEVSSVHKQQPNP 180
Qy 181 LAVSEASPTPKKNVESFGQRTSIYRGVTRHRWTGRYEAHLWDNSCRREGQSRKGRQVYLG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 LAVSEASPTPKKNVESFGQRTSIYRGVTRHRWTGRYEAHLWDNSCRREGQSRKGRQVYLG 240
Qy 241 GYDKEDKAARAYDLAALKYWGPTTTTNFPISNYESELEEMKHMTRQEFVASLRRKSSGFS 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GYDKEDKAARAYDLAALKYWGPTTTTNFPISNYESELEEMKHMTRQEFVASLRRKSSGFS 300
Qy 301 RGASMYRGVTRHHQHGRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRGLNAVT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 RGASMYRGVTRHHQHGRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRGLNAVT 360
Qy 361 NFDISRYDVKSIASCNLPVGGLMPKPSPATAAADKTVDLSPSDSPSLTTPSLTFNVATPV 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 NFDISRYDVKSIASCNLPVGGLMPKPSPATAAADKTVDLSPSDSPSLTTPSLTFNVATPV 420
Qy 421 NDHGGTFYHTGIPIKPDPADHYWSNIFGFQANPKAEMRPLANFGSDLHNPSPGYAIMPVM 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 NDHGGTFYHTGIPIKPDPADHYWSNIFGFQANPKAEMRPLANFGSDLHNPSPGYAIMPVM 480
Qy 481 QEGENNFGGSFVGSDGYNNHSAASNPVSAIPLSSTTTMSNGNEGYGGNINWINNNISSSY 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 QEGENNFGGSFVGSDGYNNHSAASNPVSAIPLSSTTTMSNGNEGYGGNINWINNNISSSY 540
Qy 541 QTAKSNLSVLHTPVFGLE 558
||||||||||||||||||
Db 541 QTAKSNLSVLHTPVFGLE 558
Alignment to SEQ ID NO: 1 to UniProt_202202 database:
RESULT 1
A0A1D6DVQ8_MAIZE
ID A0A1D6DVQ8_MAIZE Unreviewed; 712 AA.
AC A0A1D6DVQ8;
DT 30-NOV-2016, integrated into UniProtKB/TrEMBL.
DT 30-NOV-2016, sequence version 1.
DT 25-MAY-2022, entry version 31.
DE SubName: Full=AP2-like ethylene-responsive transcription factor AIL5 {ECO:0000313|EMBL:ONM12807.1};
GN ORFNames=ZEAMMB73_Zm00001d002025 {ECO:0000313|EMBL:ONM12807.1};
OS Zea mays (Maize).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; Liliopsida; Poales; Poaceae; PACMAD clade;
OC Panicoideae; Andropogonodae; Andropogoneae; Tripsacinae; Zea.
OX NCBI_TaxID=4577 {ECO:0000313|EMBL:ONM12807.1};
RN [1] {ECO:0000313|EMBL:ONM12807.1}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC TISSUE=Seedling {ECO:0000313|EMBL:ONM12807.1};
RG Maize Genome Sequencing Project;
RA Ware D.;
RT "Update maize B73 reference genome by single molecule sequencing
RT technologies.";
RL Submitted (DEC-2015) to the EMBL/GenBank/DDBJ databases.
CC -!- SUBCELLULAR LOCATION: Nucleus {ECO:0000256|ARBA:ARBA00004123}.
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DR EMBL; CM007648; ONM12807.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A1D6DVQ8; -.
DR SMR; A0A1D6DVQ8; -.
DR GO; GO:0005634; C:nucleus; IEA:UniProtKB-SubCell.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0003700; F:DNA-binding transcription factor activity; IEA:InterPro.
DR CDD; cd00018; AP2; 1.
DR Gene3D; 3.30.730.10; -; 2.
DR InterPro; IPR001471; AP2/ERF_dom.
DR InterPro; IPR036955; AP2/ERF_dom_sf.
DR InterPro; IPR016177; DNA-bd_dom_sf.
DR Pfam; PF00847; AP2; 1.
DR SMART; SM00380; AP2; 2.
DR SUPFAM; SSF54171; SSF54171; 2.
DR PROSITE; PS51032; AP2_ERF; 2.
PE 4: Predicted;
KW DNA-binding {ECO:0000256|ARBA:ARBA00023125};
KW Nucleus {ECO:0000256|ARBA:ARBA00023242};
KW Transcription {ECO:0000256|ARBA:ARBA00023163};
KW Transcription regulation {ECO:0000256|ARBA:ARBA00023015}.
SQ SEQUENCE 712 AA; 76709 MW; 931FAAF1175FB434 CRC64;
Query Match 100.0%; Score 3729; DB 80; Length 712;
Best Local Similarity 100.0%;
Matches 712; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MWAERVVGERRMRQIQRFARNAKLTVVCLLLTVVVLRGTIGAGRFGTPQQVLIELRQHFV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MWAERVVGERRMRQIQRFARNAKLTVVCLLLTVVVLRGTIGAGRFGTPQQVLIELRQHFV 60
Qy 61 SHPHRALAEHHDARSRASTTTTSSSSSSGRRDEPDPPPRSLRDPPYTLGPKISDWDEQRA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 SHPHRALAEHHDARSRASTTTTSSSSSSGRRDEPDPPPRSLRDPPYTLGPKISDWDEQRA 120
Qy 121 AWHRRHPETPPFLNDIKPRVLRDGPAHLPRPTAERGISPLFPDPRELGRTNPPPTPAGRE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 AWHRRHPETPPFLNDIKPRVLRDGPAHLPRPTAERGISPLFPDPRELGRTNPPPTPAGRE 180
Qy 181 SHEVTARVRRRFPFSPHVVVMDTSHHYHPWLNFSLAHHCDLEEEERGAAAELAAIA GAAP 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SHEVTARVRRRFPFSPHVVVMDTSHHYHPWLNFSLAHHCDLEEEERGAAAELAAIA GAAP 240
Qy 241 PPKLEDFLGGGVATGGPEAVAPAEMYDSDLKFIAAAGFLGGSAAAAATSPLSSLDQAGSK 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 PPKLEDFLGGGVATGGPEAVAPAEMYDSDLKFIAAAGFLGGSAAAAATSPLSSLDQAGSK 300
Qy 301 LALPAAAAAPAPEQRKAVDSFGQRTSIYRGVTRTKQVFGDCTGGLAGTRHICGTTAADAK 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LALPAAAAAPAPEQRKAVDSFGQRTSIYRGVTRTKQVFGDCTGGLAGTRHICGTTAADAK 360
Qy 361 GRAARAAKKHHIADGTIFFHESEGGYDKEEKAARAYDLAALKYWGSSTTTNFPVAEYEKE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 GRAARAAKKHHIADGTIFFHESEGGYDKEEKAARAYDLAALKYWGSSTTTNFPVAEYEKE 420
Qy 421 VEEMKNMTRQEFVASLRRKSSGFSRGASIYRGVTRHHQHGRWQARIGRVAGNKDLYLGTF 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 VEEMKNMTRQEFVASLRRKSSGFSRGASIYRGVTRHHQHGRWQARIGRVAGNKDLYLGTF 480
Qy 481 STEEEAAEAYDIAAIKFRGLNAVTNFEISRYNVETIMSSNLPVASMSSSAAAAAGGRSSK 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 STEEEAAEAYDIAAIKFRGLNAVTNFEISRYNVETIMSSNLPVASMSSSAAAAAGGRSSK 540
Qy 541 ALESPPSGSLDGGGGMPVVEASTAPPLFIPVKYDQQQQEYLSMLALQQHHQQQQAGNLLQ 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 ALESPPSGSLDGGGGMPVVEASTAPPLFIPVKYDQQQQEYLSMLALQQHHQQQQAGNLLQ 600
Qy 601 GPLVGFGGLYSSGVNLDFANSHGTAAPSSMAHHCYANGTASASHEHQHQMQQGGENETQP 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 GPLVGFGGLYSSGVNLDFANSHGTAAPSSMAHHCYANGTASASHEHQHQMQQGGENETQP 660
Qy 661 QPQQSSSSCSSLPFATPVAFNGSYESSITAAGPFGYSYPNVAAFQTPIYGME 712
||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 QPQQSSSSCSSLPFATPVAFNGSYESSITAAGPFGYSYPNVAAFQTPIYGME 712
Hence it would have been obvious to try choosing from a finite number of identified PLT5 genes with predictable solution for the method of modification in maize plant would be to use PLT5 gene from maize as SEQ ID NO: 1 using Biolistic Gold particles delivery system with a reasonable expectation of efficient embryogenesis as taught by Tsuwamoto et al. and Prasad and transiently introduce the PLT5 gene as taught by Tsuwamoto et al. Furthermore, it is obvious to one of ordinary skill of art before the effective filing date of the claimed invention to simply substitute the cell division promoting transcription factors (OPD2 and WUS) taught by Svitashev et al. with the known PLT5 gene of maize as SEQ ID NO: 1 and coat the gene with one or more microparticles as taught by Tsuwamoto et al. for biolistic transformation and introduce the microparticles into maize plant cell along with the transiently present and transiently active genome engineering component as RNP complex and identifying and cultivating maize plant cell without selectable marker as taught by Svitashev et al.. Furthermore, introduce the microparticles coated with TSA as taught by Bertrand et al. and further suggested by Yang et al., Horlbeck et al., and Tanaka et al., hormone for example 2, 4D as taught by Ananda et al., Joshi et al. and Samuel et al. and Spermidine et al. taught by Klein et al.
Response to Argument
Applicant's arguments filed 01/30/2026 have been fully considered but they are not persuasive.
Applicant argues none of the cited references teaches, discloses, or suggests a coating for a metal nanoparticle comprising the recited components of a genome engineering component, a second compound, and spermidine as recited in claim 1 (Response to Rejection, page 8, paragraph 3). Applicant argues Svitashev does not teach, disclose, or suggest such a coating. Applicant argues none of the other cited references cures the deficiencies of Svitashev to render the claimed invention obvious (Response to Rejection, page 8, paragraph 4).
Applicant argues Bertrand discloses polymer-based nanovectors that require release of an active ingredient by " ... the endosome/lysosome route in the reticuloendothelial system owing to the acid pH, by cleavage of the spacer El, in particular for compounds larger than 200 kDa and in particular in the case of compounds larger than 3200 kDa, or by another route for the active ingredients that are sensitive to the acid environment ... " See Bertrand, paragraph [0134]; see also Fig. 1. Applicant argues the nanovectors disclosed in Bertrand do not have a coating as recited in claim 1. Applicant argues one of ordinary skill in the art, on reading Bertrand, would not have any guidance or motivation to modify the disclosed polymer-based nanovectors with conjugated active ingredients to arrive at the claimed invention including a nonporous metal nanoparticle coated with spermidine, a genome engineering component and a second compound that are introduced by biolistic bombardment as recited in claim 1, which do not need to be released by the endosome/lysosome route as required in Bertrand (Response to Rejection, page 8, paragraph 5).
Applicant argues Brinker does not teach, disclose, or suggest the claimed coating. Applicant argues Brinker discloses mesoporous silica nanoparticles (MSNPs) that have a defined structure arranged in discrete layers, such as a central core, a supported lipid layer (shell), and additional modifiers (e.g., targeting ligands) (Response to Rejection, page 8, last paragraph). Applicant argues the MSNPs are porous (see, e.g., [0072] and [0180] of Brinker describing the size of the pores) such that the cargo, e.g., a CRISPR component and a second compound, are present either adsorbed into the pores or within the core of the MSNP. See, e.g., [0165] and [0186]-[0191] of Brinker, discussing adsorption into the pores and loading of the MSNP core. Applicant argues these paragraph also disclose that the cargo is complexed with a biological package and then this complex is encapsulated within the MSNP. Applicant argues Brinker also discloses that the cargo can be linked to a supported lipid bilayer (SLB) surrounding the porous core. See, e.g., [0177] of Brinker (Response to Rejection, page 9, first paragraph).
Applicant respectfully disagrees with Examiner's interpretation of Brinker as disclosing metal microparticles in [0150] and [0208]. See Office Action, e.g., p. 10 and 31. Applicant argues reading the entirety of [0150], this paragraph discusses how DNA is packaged for inclusion in the MSNPs:
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(Response to Rejection, page 9, paragraphs 2-3).
Applicant argues Brinker clearly discloses that the "packaged" DNA that is packaged with gold nanoparticles is present within the silica carrier, either in the pores, within the nanoporous silica core, or complexed with a biological package and encapsulated within the MSNP, not on its own as a nanoparticle. Applicant argues specifically regarding the disclosure of NanoFlares, Applicant submits that it is well known in the art that NanoFlares utilize a distinct method of attaching the oligonucleotides to the gold nanoparticle, namely via adding a 3' thiol group to ssDNA (see Chenab et al., Biomedical applications of nanoflares: Targeted intracellular fluorescence probes, Nanomedicine. 2019 April; 17: 342-358 (submitted herewith)). Applicant argues the instant application does not have such linkages between the coating and the genome engineering component (Response to Rejection, page 9, second to last paragraph).
Applicant argues Paragraph [0208] of Brinker discusses the schematic shown in Fig. l0C, replicated below, stating that. Applicant argues the nanoparticle containing the CRISPR component is clearly described as being contained within the MSNP disclosed in Brinker (Response to Rejection, page 10, paragraph 1-3).
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Applicant argues MSNPs such as those disclosed in Brinker are designed so that the silica coating around the metal nanoparticle is optimized to bind different molecules to the surface of the silica coating. Applicant argues the silica coating can be functionalized with various groups such as hydroxyl groups or PEG molecules with different end groups including amines, thiols, etc. Applicant argues the metal nanoparticle serves other purposes ( e.g., imaging) and is not used as a surface for attaching molecules (see, e.g., Homan et al. Silica-coated Gold Nanoparticles: Properties, Apps, available at https://www.sigmaaldrich.com/US/en/technical-documents/technical- article/ materials-science-and-engineering/bi osensors-and-imaging/ silica-coated-gold- nanoparticles? srsl ti d = Af mB Ooqgw J 9cq h 46 s5W l oHU n UPGb 9 F A6bjLJQqe4giEQ6soWt_ rwQ_ C, accessed on January 22, 2026, submitted herewith) (Response to Rejection, page 10, last paragraph).
Applicant argues there is no guidance or motivation in Brinker to modify the MSNPs to arrive at the coated nonporous metal nanoparticles recited in claim 1 (Response to Rejection, page 11,first paragraph).
Applicant argues the MSNPs disclosed in Brinker are taken up into the target cell via endocytosis. See, e.g., Examples 6, 8, and 13 and Figs. 1 0C and 13 of Brinker. Applicant argues this uptake process is patentably distinct from that used to deliver the claimed microparticles, which involves biolistic bombardment of the coated nonporous metal microparticles into the maize plant cells. Applicant argues One of ordinary skill in the art would not have any guidance or motivation to modify the MSNPs disclosed in Brinker to omit the targeting ligands, endosomatic peptides, and cell-penetrating peptides to be suitable for use in biolistic bombardment, as Brinker clearly discloses that these ligands and peptides are needed in order for the MSNPs to be endocytosed and subsequently exit the vesicle for cargo delivery (Response to Rejection, page 11, paragraph 2).
Applicant argues one of ordinary skill in the art, on reading Brinker, would not have any guidance or motivation to modify the disclosed MSNPs to arrive at the claimed invention including a nonporous metal nanoparticle coated with spermidine, a genome engineering component and a second compound that is introduced into the maize plant cell by biolistic bombardment as recited in claim 1. Applicant argues such a person, on reading Brinker, would understand that the MSNPs are designed to be internalized by endocytosis given the surface modifications to enhance such uptake, and then to facilitate their own release from the vesicles in order to deliver their cargo into the cell interior. Applicant argues thus, she would not have a reasonable expectation of success in modifying the MSNPs disclosed in Brinker to arrive at the claimed invention save for impermissible hindsight (Response to Rejection, page 11, paragraph 3).
Applicant argues newly cited references Samuel, Joshi, and Klein do not cure the deficiencies of Svitashev in combination with the other cited references to arrive at the claimed invention (Response to Rejection, page 11, paragraph 4).
Applicant argues Samuel is directed to non-invasive, i.e., non-biolistic bombardment, methods of introducing microparticles into plant cells. Importantly, Samuel discloses the following:
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See Samuel, col. l line 57-col. 2 line 9, emphasis added (Response to Rejection, page 11, last paragraph, page 12 first paragraph).
Applicant argues Samuel arguably teaches away from using biolistic bombardment to introduce microparticles containing proteins into plant cells. Applicant argues one of ordinary skill in the art, on reading Samuel, would not have any guidance or motivation to use biolistic bombardment to introduce a microparticle coated with, among other things, a protein such as TSA or a plant hormone, into a plant cell. Applicant argues such a person would understand that the only way to introduce such a microparticle into a plant cell is by microinjection or by the non-invasive methods developed and disclosed in Samuel. Applicant argues thus, one of ordinary skill in the art would not have a reasonable expectation of success in combining Samuel with any of Svitashev, Bertrand, Brinker, or the other cited references to arrive at the claimed method using biolistic bombardment to introduce the claimed nonporous metal microparticle into a plant cell (Response to Rejection, page 12, paragraph 2).
Applicant argues Joshi does not cure the defects of the other cited references to provide any guidance or motivation to arrive at the claimed method. Applicant argues Joshi is directed to transmucosal uptake of microparticles coated with insulin in Wistar rats. See, e.g., Abstract. Applicant argues as animal cells do not have cell walls like plant cells, Joshi does not provide a reasonable expectation of success in being able to introduce the claimed nonporous metal microparticle into a plant cell (Response to Rejection, page 12, paragraph 3).
Applicant argues Klein does not cure the defects of the other cited references to provide any guidance or motivation to arrive at the claimed method. Applicant argues Although Klein is directed to biolistic bombardment of microparticles into plant cells, only DNA (specifically plasmid DNA) is present on the microparticles. Applicant argues Klein is silent on the presence of proteins on the introduced microparticles and thus cannot provide any guidance or motivation to include proteins on the microparticles. Applicant argues one of ordinary skill in the art, on reading Klein, would not have a reasonable expectation of success in including proteins on the microparticles and biolistically bombarding them into plant cells (Response to Rejection, page 12, last paragraph).
Applicant argues as discussed in more detail above, the only references that disclose introduction of a particle containing TSA (i.e., Brinker and Bertrand) rely on endocytosis for uptake of the particle, not biolistic bombardment as required in claim 1, and Bertrand is specifically directed to uptake in animal cells. Applicant asserts Joshi discloses uptake of a microparticle coated with insulin, but in rat cells. Applicant argues as clearly stated in Samuel, it was known in the art that delivery of proteins into plant cells (i.e., cells with cell walls) on microparticles required microinjection, not biolistic bombardment or other invasive methods of introduction. Applicant argues thus, one of ordinary skill in the art, on reading the cited references, would not have a reasonable expectation of success in introducing the nonporous metal microparticle coated with spermidine, a genome engineering component and a second compound that is a protein (i.e., TSA or a phytohormone) into a maize plant cell by biolistic bombardment (Response to Rejection, page 13, first paragraph).
Applicant argues the cited references, alone or in combination, do not provide any guidance or motivation to modify the methods disclosed in Svitashev to include a nonporous metal nanoparticle coated with spermidine, a genome engineering component and a second compound, save for impermissible hindsight (Response to Rejection, page 13, paragraph 2).
Applicant argues for at least the reasons above, the teachings of Svitashev, alone or in combination with any of the other cited references, fail to render the claimed invention obvious (Response to Rejection, page 13, paragraph 3).
Applicant's arguments have been fully considered but they are not persuasive, since:
Regarding argument on Bertrand does not teach metal microparticles, the argument was not found persuasive since Brinker et al. teaches a Mesoporous silica nanoparticles (MSN) comprising a metal oxide core and a CRIPSR component as cargo (claim 1), a guide component, a nuclease (claim 6) and further comprising a trichostatin A (TSA) (claims 12 and 15). Brinker et al. teaches packaging DNA with gold nanoparticles (page20, paragraph 0150). Brinker et al. teaches a complex comprising a nanoparticles e.g. Gold or metal nanoparticles that is used to package the DNA. Therefore the use of metal microparticles coatings for transfer of TSA, CRISPR components and hormones are explicitly taught by Brinker et al. Furthermore, Svitashev et al., Joshi et al., Brinker et al., Klein et al. expressly teaches a metal microparticle delivered into cell comprising TSA, hormones CRISPR complex and Spermidine, which would have predictable results of developing a method of generic medication in maize.
Regarding argument on Brinker teaches MSNPs are porous, the argument was not found persuasive since Brinker teaches a particle can have variety of shapes and cross-sectional geometries that may depend, in part, upon the process used to produce the particle e.g. spherical nanoparticles (page 8, paragraph 0068). Brinker teaches use of gold nanoparticles for packaging (page 20, 0150, page 26, paragraph 0208). Furthermore, Brinker teaches confinement of package including DNA within nanoporous silica core (page 20, paragraph 0150) and the silica shell could be non-porous (page 30, paragraph 0246). Therefore if the package would have been done in nonporous silica it would have been obvious to use gold particles that are nonporous. Furthermore, Svitashev et al. teaches use of gold particles which would be non-porous coated with RNP/DNA which were loaded onto macrocarrier and that were allowed to dry and the immature embryo was bombarded with the particle using a PDS-1000/He Gun (Bio-Rad, USA). For example Wang et al. (Published: 12/20/2017, Book: Noble and Previous Metals-Properties, Nanoscale effects and applications, Chapter: Applications of Gold Nanoparticles in Cancer Imaging and Treatment, IntechtOpen, http://dx.doi.org/10.5772/intechopen.70901) teaches given the strong binding affinity of gold to thiol and amine groups, the surface of gold nanoparticles can be easily functionalized with biomolecules such as DNA, siRNA, peptides, antibodies, and receptors (page 292, first paragraph).
Furthermore, TSA has been used as an experimental drug for example as an antifungal agent (Tsuji et al. (Published: 1974, Journal: The Journal of Antibiotics 49-14691 pages 1-6). Therefore someone skilled in the art would confine the hormones, TSA, CRISPR components and Spermidin so the one or more composition would be confined to the metal microparticles that would be non-porous leading to the invention of the method of genetic modification.
Furthermore, Parveen et al. (Published: 2012, Journal: Nanomedicine: Nanotechnology, Biology, and Medicine 8:147-166) teaches metal based NPs can be synthesize in extremely small sizes and thus the large surface area provides the ability to carry a relatively higher dose of drugs (page 157, last paragraph). Furthermore, Joshi et al. teaches gold nanoparticles are successfully used for transmucosal insulin hormone delivery (page 301, Abstract, Scheme 1). Joshi et al. teaches nanogold- insulin would permeate in mucosal cells (page 304, left last paragraph). Joshi et al. teaches Nanoparticles could also serve as excellent delivery vehicles for a variety of biomolecules such as proteins, DNA, and drugs (page 300, left paragraph 1). Joshi et al teaches tunable shape- and size-dependent optical properties of gold nanoparticles have been exploited in various surface coatings and biomedical applications which are biocompatible, nontoxic, bind readily to a large range of biomolecules such as amino acids, proteins/enzymes, and DNA, and expose large surface areas for the immobilization of such biomolecules (page 300, left paragraph 2). Joshi et al. teaches the ability to modulate the surface chemistry of gold nanoparticles by binding suitable ligands has important applications in many areas such as novel organic reactions, sensors (both inorganic and biological entities), drug/DNA delivery, and imaging (page 300, right paragraph1). Therefore, it would have been obvious to use metal microparticles such as nonporous gold microparticles to deliver CRISPR components, phytohormones and TSA.
Regarding Klein et al. does not teach other proteins on the introduced microparticles, the argument was not found persuasive since based on claim 1 the components as CRISPR components, TSA, phytohormones and spermidine would be present in one or more microparticles and Klein et al. teaches one of the microparticle coated with DNA and spermidine. Klein et al. teaches spermidine that was used to precipitate or adsorption of DNA to the surface of the microprojectiles and used for condensing materials that would be less susceptible to degradation by intracellular nucleases, influenced the efficiency of DNA delivery and increased expression units (page 560, right paragraph 3 and page 561 paragraphs 1-2). Therefore, someone skilled in the art would use the microparticle in the method of genetic modification involving introducing microparticle coated with CRISPR components, TSA and phytohormones.
The advantage of co-transferring the components was predictable given the prior arts described above. Given the RNPs were successfully transferred in monocot maize by Svitashev et al. and furthermore Tsuwamoto et al.’s teachings of particle delivery of a boost gene as PLT5 gene and the teachings of nanoparticle with TSA of Bertrand et al. and further suggestions by Yang et al., Horlbeck et al., and Tanaka et al. to use these chemicals to improve the regeneration of the genetically modified plant was predictable that would lead to the claimed method of genetic modification in maize plant cell. Since the combined transfer of a nanoparticle comprising CRISPR component, TSA and hormone was known, utilization of technique in a maize plant would not have been surprising since such transfer using microparticle for the CRISPR components was known in the art by Svitashev et al.
The rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art, established scientific principles.
Furthermore, the strongest rationale for combining references is a recognition, expressly or impliedly in the prior art or drawn from a convincing line of reasoning based on established scientific principles or legal precedent, that some advantage or expected beneficial result would have been produced by their combination, hence such advantage was obvious over the teaching of prior arts.
Furthermore, where a rejection of a claim is based on two or more references, a reply that is limited to what a subset of the applied references teaches or fails to teach, or that fails to address the combined teaching of the applied references may be considered to be an argument that attacks the reference(s) individually. Where an applicant’s reply establishes that each of the applied references fails to teach a limitation and addresses the combined teachings and/or suggestions of the applied prior art, the reply as a whole does not attack the references individually as the phrase is used in Keller and reliance on Keller would not be appropriate. This is because the test for obviousness is what the combined teachings of the references would have suggested to a person having ordinary skill in the art (PHOSITA).”
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.
Nonstatutory double patenting over copending Application No. 18740126 in view Svitashev et al., Tsuwamoto et al., Prasad et al. Horlbeck et al., Yang et al., Tanaka et al., and Klein et al.
Claims 1, 4-6, 13 and 16 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 34-36, 42-47 of copending Application No. 18/740,126 in view of Svitashev et al., Tsuwamoto et al., Prasad et al. Horlbeck et al., Yang et al., Tanaka et al. and Klein et al.
Regarding claims 1, 4-6, and 16 see analysis above with regard to obviousness over Svitashev et al., Tsuwamoto et al., Prasad et al., Horlbeck et al., Yang et al., Tanaka et al. to develop a method of using particle coated with genetic engineering component, TSA and SEQ ID NO: 1 and 2 for more efficient regeneration of the genetically modified maize plant.
Furthermore, Copending Application No. ‘126 claims 34-35 teaches a microparticle coated with a coating comprising a genomic engineering component.
Copending Application No. ‘126 claims 42-44 teaches the microparticle is further coated with an epigenetically regulating chemical, a histone deacetylease inhibitor (HDACi), a trichostatin A (TSA).
Copending Application No. ‘126 claims 46-47 teaches the microparticle is further coated with phytohormone which is 2, 4 D, 6-Benzylaminopurine and Zeatin.
Copending Application No. ‘126 claim 36 teaches a kit for the genetic modification of a plant genome by microprojectile (i.e. biolistic) bombardment which is a means for coating their disclosed microparticles.
Furthermore, Svitashev et al. teaches the gold (i.e. metal) microparticles (0.6 µm) coated with RNA RNP complex, helper genes-cell division promoting transcription factors were introduced into the maize embryo cells via biolistic bombardment using helium gene gun (page 2, right paragraphs 2-3, page 5, right last paragraph).
Thus someone skilled in the art would use the technique of copending Application No. ‘126 to improve method developed by Svitashev for biolistic introduction of nonporous gold microparticles coated with RNP/DNA complex for higher efficiency of genome engineering by adding the TSA and phytohormones as 2, 4 D, 6-Benzylaminopurine and Zeatin.
Copending Application No. ‘126 does not teach their method comprise introducing Spermidine.
Klein et al. teaches spermidine that was used to precipitate or adsorption of DNA to the surface of the microprojectiles and used for condensing materials that would be less susceptible to degradation by intracellular nucleases, influenced the efficiency of DNA delivery and increased expression units ( page 560, right paragraph3 and page 561 paragraphs 1-2). Klein et al. teaches using 1.2 µm microprojectile (page 559, right last paragraph). Klein et al. teaches the suspension cells were bombarded with tungsten particles coated with DNA and Spermidine (page563, left paragraph 2). Therefore the coating comprising Spermidine would have been the routine laboratory molecule for efficient delivery of the DNA using microparticles.
Therefore it would have been obvious to someone skilled in the art before the effective date of filing to develop a method of genetic modification comprising introducing a macroparticle coated with CRISPR component, TSA and a phytohormone which is 2, 4 D, 6-Benzylaminopurine and Zeatin and furthermore the method would comprise introducing Spermidine in the metal microparticles as taught by Klein et al. leading to the method that would have that efficient delivery of the metal microparticles.
Regarding claim 13, Svitashev et al. teaches a method for producing a genetically modified maize plant, comprising the steps of genetically modifying a maize plant cell by co-introducing RNP and maize ovule development protein and maize Wuschel protein (page 5, right paragraph 4, Methods) and regenerating a plant from the modified cell (page 5, right paragraph 7). Furthermore, Svitashev et al. teaches the genetic analysis of their regenerated T0 generation with expected mendelian ratio of 1:1 further showed the activity of genome engineering component were transient (page 4, right paragraph 2).
The method of producing genetically modified plant by modifying a maize plant cell, regenerating maize plant that does not contain any of the genome engineering component and second compound for example TSA used for co-introduction in the cell would not stably transfer to next generation and application of cointroduction of genome engineering component with TSA would be obvious over the technique from Copending Application No. ‘126.
Furthermore, the treatment with the mass of 15 ng to 45 ng Trichostatin A (TSA), and 125 ng to 500 ng of a phytohormone selected from 2,4-Dichlorophenoxyacetic acid, 6-benzylaminopurine, zeatin, and combinations thereof, would have been obvious to try for microparticles with size of the microparticle in a range of 0.4-1.6 micron (μm), preferably 0.4-1.0 μm (Spec, page 30, paragraph 3), such small microparticle also showed by Klein et al. with microparticle diameter size of 0.6, 1.2 μm (page 561, right paragraph 2). It would have been obvious from the finite number of identified, predictable solutions, with a reasonable expectation of finding the specific mass range of TSA and the phytohormones for treatment using the microparticles in the method of genetic modification in a maize plant cell.
This is a provisional nonstatutory double patenting rejection.
Response to Argument
Applicant's arguments filed 01/30/2026 have been fully considered but they are not persuasive.
Applicant submits that the amendments to claim 1 presented herein render the rejection moot for at least the reasons discussed in more detail above and requests the rejection of double patenting be withdrawn.
Applicants’ arguments are fully considered but they are not persuasive since the amendment stating the, introducing “nonporous” metal microparticles and introducing one of more the components via “biolistic bombardment” would have been obvious over Svitashev et al. in the method of genetic modification in a maize plant cell, see analysis above in view of copending Application No. 18/740,126, see analysis above.
Summary
No claim is allowed.
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/SANTOSH SHARMA/ Examiner, Art Unit 1663
/DAVID H KRUSE/Primary Examiner, Art Unit 1663