DETAILED ACTION
Claims 1-2, 5-7, and 9-22 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
The rejection of claims 8-10 and 13-14 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter that the inventor or a joint inventor, or for pre-AIA the applicant, regards as the invention is withdrawn in light of Applicant’s amendment to the claims.
Claim Objections
Claims 1 and 6 are objected to because of the following informalities:
In the last line of claims 1 and 6 “a” should be replaced with --the--.
Applicant’s amendment to the claims overcame the claim objections set forth in the Office action mailed 11 March 2025.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 7-9 and 12 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
The rejection is repeated for the reasons of record as set forth in the Office action mailed 11 March 202. Applicant’s arguments filed 11 August 2025 have been fully considered but they are not persuasive.
Parent claim 11 is drawn to a method comprising introducing the nucleic acid of claim 1 into a plant cell and selecting a plant cell where the nucleic acid is integrated into a plant organellar genome or where the nucleic acid is an independent replicon in an organelle.
Dependent claim 12 recites that the organellar genome is a mitochondrial or plastid genome. However, there are no other organelles in plant cells that have genomes and no other organelles that can support replication. Thus, the claim fails to further limit the subject matter of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Response to Arguments
Applicant urges that the claims were amended (response pg 8).
This is not found persuasive because no amendment was made to overcome the rejection over claim 12. Applicant’s amendment to the claims overcame the previous rejection over claims 7-9.
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 1-2, 5-7, and 9-22 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for methods of expressing proteins from mini-chromosomes located in plastids of tobacco cells that also express a viral replicase (Rep) protein, does not reasonably provide enablement for methods of expressing proteins from mini-chromosomes located in in plastids of cells that lack a Rep protein or for stable organellar genome transformation in the full scope of plant species. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Due to Applicant’s amendment of the claims, the rejection is modified from the rejection set forth in the Office action mailed 11 March 2025. Applicant’s arguments filed 11 August 2025 have been fully considered but they are not persuasive.
A. The specification does not teach how to make the full scope of claimed plants whose organelles are transformed with a polynucleotide comprising an organellar transgene cassette comprising an origin of replication derived from a geminivirus, a flanking sequence, a transgene of interest, another flanking sequence, and another origin of replication derived from a geminivirus.
One embodiment of the claims is drawn to a polynucleotide comprising an organellar transgene cassette comprising an origin of replication derived from a geminivirus, a flanking sequence, a transgene of interest, another flanking sequence, and another origin of replication derived from a geminivirus, methods of transforming organelles of plant cells and plants with the polynucleotide, and plant cells and plants where the polynucleotide is stably integrated into the organellar genome. This polynucleotide is the one recited in claim 1, part (i), and will be referred to as polynucleotide 1(i) in this rejection.
Polynucleotide 1(i) has the viral origins of replication outside the flanking sequences. When polynucleotide 1(i) is transformed into plastids, homologous recombination will insert the transgene of interest between the plastid genomic regions delineated by the flanking sequences and the viral origins of replication will be eliminated along with the rest of the vector on which the polynucleotide is located.
The only working example of plants transformed with a polynucleotide comprising an organellar transgene cassette comprising an origin of replication derived from a geminivirus, a flanking sequence, a transgene of interest, another flanking sequence, and another origin of replication derived from a geminivirus is Agrobacterium-mediated transformation of tobacco plastids with co-transformation with a nuclear construct encoding the Rep gene corresponding to the viral origin of replication (pg 66, line 18, to pg 67, line 18; pg 73, line 21, to pg 74, line 11). The plastid transformation constructs also contained the aadA selectable marker (figure 19), and Agrobacterium-mediated plastid transformation of tobacco requires selection on spectinomycin (pg 73, lines 17-20).
The instant specification fails to provide guidance for how to make the full scope of plants with stably transformed organellar genomes over the full scope of the claims.
A1. The specification does not teach how to transform the plastids of the full scope of claimed plants with polynucleotide 1(i)
Claims 9-10 and 21-22 recite that the plant is one of Nicotiana, potato, maize, canola, Brassica, rice, wheat, barley, cotton, algae, lemnospora, moss, tomato, capsicum, squash, sunflower, soybean, carrot, melons, grape, lettuce, strawberry, sugar beet, pea, or sorghum.
While most of the recited plants are individual species, there are approximately 12,000 moss species and 100,000 or more algal species.
The specification does not provide guidance for how to transform chloroplasts the full scope of claimed plant species.
Even though plastid transformation was first developed 30 years prior to the filing of the instant application, it has only been successful in a very few plant species and in no monocots (Bock, 2015, Annual Review of Plant Biology 66:211-241, see pg 221, ¶4; Table 2; Maliga, 2022, Nature Plants 8:996-1006, see pg 996, right column, ¶2, to pg 997, left column, ¶1, Table 1). Plastid transformation is not possible even in many plant species where nuclear transformation is well-established (Jakubiec et al, 2021, Nature Plants 7:932–941, see pg 937, right column, ¶4).
DNA delivery methods that work in species like Nicotiana benthamiana do not work in other plant species. For example, successful plastid transformation in Arabidopsis required development of new plastid transformation methods (Maliga, pg 997, left column, ¶1) and was not achieved until after the filing of the instant priority document (Maliga, pg 1003, right column, ¶3, which cites reference 77, published in 2021). Development of such new methods requires undue trial and error experimentation.
Of the plants claimed, wheat, barley, cotton, squash, sunflower, melons, grape, strawberry, pea, or sorghum are not on the lists provided by Bock and Maliga. Plastid transformation is only enabled in two (Physcomitrella patens and Marchantia polymorpha) of the approximately 12,000 moss species and one (Chlamydomonas reinhardtii) of the approximately 100,000 algal species. The specification does not teach how to transform the plastids of these plant species.
Insertion of genes into the chloroplast relies on homologous recombination, which requires that the vectors comprising a desired transgene contain flanking sequences that have close to 100% homology to the sequences flanking the desired insertion site in the chloroplast genome (Bock, pg 218, ¶3; pg 223, ¶3; Figure 4). As such, the sequence of regions flanking the desired integration site in the chloroplast genome must be known (Bock, pg 218, ¶3; pg 223, ¶3; Figure 4).
Even slight differences between the regions flanking the target site in the chloroplast genome and those on the designed nucleic acid vector for transformation result in considerably reduced or eliminated transformation efficiency (Daniell et al, 2016, Genome Biology 17:1-29; paragraph spanning the columns on pg 12). As such, one of skill in the art is often required to utilize species-specific flanking regions with 100% identity to those in the desired chloroplast genome (Daniell et al, pg 12, left column ¶4, to right column, ¶2).
As of September 2023 (after the time of filing), fewer than 13000 chloroplast genomes were sequenced in plants (Wang et al, 2024, Trends Plant Sci. 29:754-769; DOI: 10.1016/j.tplants.2023.12.014; pg 2 ¶2; Figure 1). Therefore, the chloroplast genomes of the full scope of claimed species are not known, making chloroplast genetic engineering through homologous recombination ineffective or impossible.
The specification does not provide sufficient teachings regarding the chloroplast genomes of the claimed species, and thus fails to provide enabling guidance for transforming the chloroplasts of the full scope of the claimed species.
Creation of transplastomic plants also requires selection of transformed cells using a suitable selectable marker to create plants in which all of the chloroplasts harbor the desired transgene, i.e., “homoplasmic plants” (Bock, Table 1; Figures 3-4; pg. 218, ¶1 to pg. 219, ¶2; pg 222, ¶2). Because of this repeated selection of cultured cells, a suitable culturing and regeneration procedure for a given plant species is also required to create a transplastomic plant (Bock, pg 218, ¶4 to pg. 219, ¶2; pg. 221, ¶4, to pg 222, ¶3).
Although each plant cell has about 1000 copies of the plastid genome, only one genomic copy is altered in the initial transformation event (Bock, pg 218, ¶4). As such, transformed cells must undergo a long process involving multiple rounds of selection and plant regeneration for homoplasmy to be achieved, i.e., for all plastids genomes to have the transformed genes and for transformation to be stable (Bock, paragraph spanning pg 218-219).
The most common selectable marker is the aadA gene, which confers resistance to spectinomycin (Bock, Table 1; pg 218, ¶1). However, not all plants are sufficiently sensitive to spectinomycin for effective selection, and cereals are not sensitive to it at all (Bock, pg 222, ¶2).
As such, extending plastid transformation to other plant species, including those claimed, will require the development of new selectable markers (Bock, pg 222, ¶2-3). Further, these selectable markers must be plastid-specific and must not target nuclear transcription or cytoplasmic translation (Bock, pg 218, ¶1).
The specification does not provide guidance for which selectable markers are applicable to the full scope of claimed plants or which selectable markers would be ineffective for selecting transplastomic cells. The specification does not provide guidance for how to conduct tissue culture and selection for the full scope of claimed plants. Finally, the specification does not provide sufficient teachings regarding the chloroplast genomes of the claimed plants species to enable one having skill in the art to develop vectors containing sequences with sufficient homology to the respective chloroplast genomes to allow for integration through homologous recombination in chloroplasts of the full scope of claimed plants.
While the specification lists selectable markers for selection of transgenic plants (pg 11, lines 24-29), it does not teach selectable markers that are effective for selection of transplastomic cells over the full scope of claimed plant species. Development of these new selectable markers and selection procedures for the full scope of claimed plant species would require undue trial and error experimentation.
Further, none of the claims require a selectable marker be in the polynucleotide. The specification does not teach how to transform plastids with a construct that does not comprise a selectable marker.
Plastid transformation also requires specialized tissue culturing and regeneration procedures (Bock, pg 221, ¶6, to pg 219, ¶2 to pg 222, ¶3). Procedures for culturing and regenerating transplastomic plants are limited to plants which have robust tissue culturing and regeneration methods available (Bock, pg 221, ¶4 to pg 222, ¶3), but the availability of such methods still is not enough for achieving plastid transformation is some plant species. For example, the procedures that work for nuclear transformation of cereals are counter-indicated for plastid transformation (Bock; pg 222, ¶2).
The specification does not provide guidance regarding culturing protocols that can be used for the full scope of claimed plants and does not provide teachings regarding modifications to the disclosed methods that could extend its use to the full scope of species encompassed by the claims. Extending methods applicable to the current list of plant species for which plastid transformation works to other plant species, including the full scope of those instantly claimed, would require “optimization work [that] is exceedingly laborious and time consuming and is based largely on the trial-and-error principle” (Bock, pg 222, ¶3).
Bock teaches that developing plastid transformation in other plants species is not routine or predictable and calls it a “daunting task” (pg 231, ¶4):
From many fruitless efforts made in the past, the community has come to realize that developing workable plastid transformation protocols for new species represents a daunting task and requires long-term investments in tedious optimization work directed toward the improvement of transformation protocols, tissue culture procedures, and selection conditions. Both the academic and the industrial sectors seem to largely shy away from making these investments.
Thus, extensive teachings are required for transforming the plastids of the full scope of claimed plants. These teachings are not provided for by the specification.
The instant specification fails to provide guidance for the plastid genomic sequences that would be required to achieve homologous recombination necessary for stable plastid transformation. The instant specification fails to provide guidance for the DNA delivery methods that would be necessary for plastid transformation in the full scope of claimed plant species. The instant specification fails to provide guidance for the selectable markers and selection procedures that would be necessary for plastid transformation in the full scope of claimed plant species. The instant specification fails to provide guidance for the tissue culturing and regeneration procedures that would be necessary for plastid transformation in the full scope of claimed plant species.
The specification also fails to overcome the unpredictability in the art as it provides no working examples of the full scope of claimed plant species whose plastids have been transformed.
Given the claim breath, nature of the invention, the state of the prior art, the level of one of ordinary skill, the unpredictability in the art, the lack of guidance in the specification, the lack of working examples over the full scope of the claims, and the undue experimentation that would be needed to make the invention based on the content of the disclosure, as discussed above, transformation of plant plastids with polynucleotide 1(i) is not enabled throughout the full scope of the claims.
A2. The specification does not teach how to transform the mitochondria with polynucleotide 1(i)
The specification provides no guidance for how to transform the mitochondria of any plant species with polynucleotide 1(i) and provides no working examples of how to achieve this.
Stable transformation of mitochondrial genomes is unpredictable due to extensive recombination in the mitochondrial genome, as well as the large size of genome, lack of conserved sequences, and variations in the stoichiometry of genes within mitochondria that exist even within the same cell (Wang et al, pg 9, ¶2, pg 10, ¶1). As result, stable mitochondrial transformation has not been achieved in any plant species, even years after the effective filing date of the instant invention (Wang et al, pg 10, ¶1).
There is no teaching of selection conditions, regeneration protocols, or the sequences of flanking regions for transformation of the mitochondria with the polynucleotide of even one plant species, much less the full scope of those claimed.
Further, none of the claims require a selectable marker be in the polynucleotide. The specification does not teach how to transform mitochondria with a construct that does not comprise a selectable marker.
Given the claim breath, nature of the invention, the level of one of ordinary skill, the lack of guidance in the specification, the lack of working examples, and the undue experimentation that would be needed to make the invention based on the content of the disclosure, transformation of plant mitochondria with polynucleotide 1(i) is not enabled.
The specification does not teach how to make the full scope of claimed plants whose organelles are transformed with a polynucleotide comprising an organellar transgene cassette comprising an origin of replication derived from a geminivirus, a transgene of interest, and another origin of replication derived from a geminivirus.
One embodiment of the claims is drawn to a polynucleotide comprising an organellar transgene cassette comprising an origin of replication derived from a geminivirus, a transgene of interest, and another origin of replication derived from a geminivirus, methods of transforming organelles of plant cells and plants with the polynucleotide, and plant cells and plants whose organelles are transformed with the polynucleotide. This polynucleotide is the one recited in claim 1, part (ii), and will be referred to as polynucleotide 1(ii) in this rejection.
In this embodiment, transformation of polynucleotide 1(ii) into plant organelles results in the formation of an autonomously replicating minichromosome.
The specification describes the transformation of tobacco, potato and maize plastids and tobacco mitochondria with the polynucleotide (pg 67, lines 13-18; pg 74, lines 12-21; pg 75, line 30, to pg 76, line 13). The methods all use a Rep gene that corresponds to the geminivirus origin of replication be present in organelle, either by cotransformation or by nuclear transformation with a construct that links the REP protein to an organellar transit peptide.
Post-filing art teaches selection with a selectable marker is necessary in the early stages of such transformation of tobacco plastids (Jakubiec et al, see, for example, the paragraph spanning the columns on pg 933; the paragraph spanning pg. 934-935; pg 935, right column, ¶2). The specification fails to teach how to transform plastids with the polynucleotide in the absence of a selectable marker.
Issues regarding the need for development of new selectable markers and selection procedures for the full scope of claimed plant species detailed above apply here as well and would require undue trial and error experimentation.
The specification fails to teach transformation of polynucleotide 1(ii) into plastids of any plant other than tobacco, potato and maize or transformation of the polynucleotide into mitochondria of any plant other than tobacco (pg 67, lines 13-18; pg 74, lines 12-21; pg 75, line 30, to pg 76, line 13). Jakubiec et al teaches that extension of this method other organisms will require will require further development (paragraph spanning pg 937-938).
Lastly, the claims do not require that the two origins of replication be from the same geminivirus. As replication of the construct requires the interaction of the rep proteins with a specific sequence in the viral origin of replication (Jakubiec et al, paragraph spanning the columns on pg 932), the REP protein needs to interact with both viral origin of replications on the polynucleotide. The specification does not teach how to make autonomously replicating chromosomes with two different origins of replication.
Thus, extensive teachings are required for one of ordinary skill in the art to make and use geminiviral vectors which are suitable for use in transforming plastids of a plant in which viral Rep proteins are absent, in the absence of a selectable marker, and for the full scope of claimed plants.
Given the claim breath, teachings in the art, nature of the invention, the level of one of ordinary skill, the lack of guidance in the specification, the lack of working examples, and the undue experimentation that would be needed to make the invention based on the content of the disclosure, transformation of plant organelles with polynucleotide 1(ii) is not enabled.
Response to Arguments
Applicant urges that they engaged in industrial collaborations involving: chloroplast-based production of double-stranded RNA in soybean; protein expression in carrot; and the production of antimicrobial proteins in Spirodela (duckweed, a monocot) for cosmetic applications (response pg 8-9).
This is not found persuasive. There are no working examples in the specification of transformation of the chloroplasts of these plants with polynucleotide 1(i) or 1(ii) and Applicant provided no evidence of successful transformation of these plants using the method as claimed. Further, even if Applicant had done so, that does not enable transformation of the full scope of claimed plants for the reasons detailed in the rejection above.
Applicant has provided no evidence that carrot, soybean, and lemna have all been transformed according to the present invention. “The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Applicant is reminded that evidence submitted after final will not be considered unless Applicant provides a showing of good and sufficient reasons of why they were necessary and not earlier presented.
Applicant urges that one of ordinary skill in the art would know that particle bombardment is the standard and primary method for delivering DNA into chloroplasts, as is its optimization of particle sizes and rupture discs (response pg 9).
This is not found persuasive. Applicant has not provided any support for such assertions. The rejection provides evidence that plastid transformation because has only been successful in a very few plant species (Bock, 2015, Annual Review of Plant Biology 66:211-241, see pg 221, ¶4; Table 2; Maliga, 2022, Nature Plants 8:996-1006, see pg 996, right column, ¶2, to pg 997, left column, ¶1, Table 1) and is not possible even in many plant species where nuclear transformation is well-established (Jakubiec et al, 2021, Nature Plants 7:932–941, see pg 937, right column, ¶4). Note that three of the authors (Jakubiec, Malcuit, and Sorokin) on post-filing reference Jakubiec et al are inventors on the instant application.
Applicant urges that since chloroplast size can vary between crop species, the preferred conditions are 0.4 or 0.6 um particles combined with 1100 or 1300 psi rupture discs, as these are less damaging to plant tissues with a GUS assay performed four days after bombardment is used to assess efficiency; this is a standard, well-established procedure for optimizing DNA delivery into plant cells, can be completed within five days, and the methodology is thoroughly documented in numerous academic publications (response pg 10).
This is not found persuasive. Applicant has not provided any of these numerous academic publications to back up these mere arguments of counsel. Applicant has also not provided evidence that such optimization would work over the full scope of claimed plant species, given the failure discussed in Bock, 2015, Maliga, 2022, and Jakubiec et al, 2021.
Applicant urges that the ballistic method for DNA delivery into chloroplasts is well established and known to the skilled person and optimizing DNA delivery into the organelles of specific crop species is a simple, efficient, and routine procedure for the skilled person (response pg 10).
This is not found persuasive. The rejection provided evidence that DNA delivery methods that work in species like Nicotiana benthamiana do not work in other plant species; successful plastid transformation in Arabidopsis required development of new plastid transformation methods (Maliga, pg 997, left column, ¶1) and was not achieved until after the filing of the instant priority document (Maliga, pg 1003, right column, ¶3, which cites reference 77, published in 2021). Applicant has not provided evidence to counter the evidence in the rejection.
Applicant urges that a small amount of DNA of the presently claimed invention delivered into the chloroplast can be rapidly amplified to optimal copy numbers, enabling both insertional and non-insertional expression; low DNA delivery is overcome by the present minichromosome-based amplification system, which provides a sufficient amount of transgene DNA for both homologous recombination and/or episomal expression and allows for efficient transformation, accelerated generation of homoplasmic plants, and stable replication and expression of non-integrative minichromosomes (response pg 10).
This is not found persuasive. Applicant has not provided evidence that their method works in the full scope of plant species or works without a selectable marker. Note that dependent claim 9 encompasses over 100,000 plant and algal species; the claims that do not require a particular species encompass a plant of any species.
Applicant urges that trnI-trnA and rbcL-accD regions have been extensively used in the art as homologous recombination sequences for plastid transformation in tobacco and several other crops; the high degree of sequence conservation in these regions across many species, it is standard practice for a person skilled in the art to design primers targeting these homologous regions to amplify corresponding fragments for use as left and right flanking sequences (LFS or RFS), followed by sequencing and incorporation into vector construction (response pg 11).
This is not found persuasive. Sequencing the plastid genomes of the full scope of plants, including that of the approximately 12,000 moss species and approximately 100,000 algal species encompassed by dependent claim 9, and finding suitable regions for transgene integration (Daniell et al, pg 12, right column, ¶2) will require undue experimentation.
Applicant urges that the presently claimed methods enable transformation of organelles without the need for flanking sequences for homologous recombination; by delivering the Rep gene along with an expression vector containing the gene of interest, circularization of the expression vector is achieved through viral origins of replication, forming a “minichromosome” that replicates and expresses independently of the chloroplast genome (response pg 11).
This is not found persuasive. The method of claim 11 requires that the DNA of claim 1 be integrated into a plant organellar genome (part 3), and the DNA of claim 1, part i), requires flanking sequences. Integration into plastid genomes requires homologous recombination. See also claim 14, which requires integration into the organellar genome.
Applicant urges that this non-integrative minichromosome approach allows for the delivery of biosynthetic pathways comprising up to 11 genes (10-15 kb), enabling the production complex molecules that are typically not feasible using traditional chloroplast transformation techniques; because the minichromosome can replicate autonomously in chloroplasts at high copy numbers, the method eliminates the need to identify and utilize optimal insertion sites (response pg 11-12).
This is not found persuasive. The method of claim 11 requires that the DNA of claim 1 be integrated into a plant organellar genome (part 3), and the DNA of claim 1, part i), requires flanking sequences. Integration into plastid genomes requires homologous recombination. See also claim 14, which requires integration into the organellar genome.
Applicant has also not provided support for the assertions that pathways comprising up to 11 genes can be delivered. “The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Applicant urges that both vectors with and without homologous flanking sequences can be successfully used according to the presently claimed invention; use of a replicative minichromosome vector with homologous sequences offers distinct advantages over traditional plastid transformation systems, including significantly higher transformation efficiency (as demonstrated by a fivefold increase in Applicant’s tobacco studies) and the ability to achieve a homoplasmic state within a single generation, in distinct contrast to the three to four rounds of selection typically required by conventional approaches to achieve a similar homoplasmic state (response pg 12).
This is not found persuasive. The specification provides no data on this alleged significantly higher transformation efficiency or ability to achieve a homoplasmic state within a single generation. Applicant has not provided a Declaration to support these mere arguments of counsel.
Applicant urges that the selection of flanking sequences for homologous recombination is a simple and routine technique for a person of ordinary skill in the art, due to the availability of previously identified insertion sites and the high degree of sequence conservation in those regions across species (response pg 12).
This is not found persuasive. Applicant has not provided evidence that the full scope of 100,000 plastid and 100,00 mitochondrial genomes encompassed by dependent claim 9 or the millions of plastid and mitochondrial genomes encompassed by “plant” have a high degree of sequence conservation. “The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Further, insertion of genes into the chloroplast relies on homologous recombination, which requires that the vectors comprising a desired transgene contain flanking sequences that have close to 100% homology to the sequences flanking the desired insertion site in the chloroplast genome (Bock, pg 218, ¶3; pg 223, ¶3; Figure 4). Even slight differences between the regions flanking the target site in the chloroplast genome and those on the designed nucleic acid vector for transformation result in considerably reduced or eliminated transformation efficiency (Daniell, paragraph spanning the columns on pg 12).
As such, selection of flanking sequences for homologous recombination the full scope of 100,00 plastid and 100,00 mitochondrial genomes encompassed by the claims is not a “simple and routine technique for a person of ordinary skill in the art”.
Applicant urges that one of ordinary skill in the art would appreciate that several universal selectable markers are available for chloroplast transformation across a range of crops; no natural resistance to nptII has been reported in any crop species (response pg 12-13).
This is not found persuasive. Applicant has provided no evidence for their assertions regarding natural resistance to nptII. “The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Applicant urges that transformation of dicots was successful using kanamycin, and monocots using gentamicin selection; they successfully utilized the nptII selectable marker gene for chloroplast transformation in soybean and carrot using kanamycin selection, and in Spirodela (a monocot species) using gentamicin selection (response pg 13).
This is not found persuasive. The specification provides no teaching of the use of gentamicin selection in Spirodela chloroplast transformation and has provided no evidence of Spirodela chloroplast transformation at all, and Applicant has provided no evidence to back up these mere arguments of counsel.
Applicant has also not provided evidence that these selectable markers would work for transformation of plastids over the full scope of 100,000 plant and algal species. The specification does not provide guidance for which selectable markers are effective for selection of transformed plastids of the full scope of claimed plants.
Bock teaches that extending plastid transformation to other plant species, including those claimed, will require the development of new selectable markers and that this is amongst the “optimization work [that] is exceedingly laborious and time consuming and … based largely on the trial-and-error principle” (Bock, pg 222 ¶2-3). Applicant does not provide teachings to overcome this over the full scope of plants.
Further, none of the claims require a selectable marker be in the polynucleotide. The plastid transformation constructs contained the aadA selectable marker (Figure 19), and plastid transformation of tobacco requires selection on spectinomycin (pg 73, lines 17-20). The specification does not teach how to transform plastids with a construct that does not comprise a selectable marker.
Applicant urges that other marker genes, such as ALS and PPO, have also been used for chloroplast transformation using herbicide selection, citing US20230313212A1; the aadA, ALS, and PPO1 selectable markers for insertion of the Rep cassette into the chloroplast genome and nptII for selection of the minichromosome vector is sufficient. The present applicant has demonstrated successful use of a mutated PPO1 gene for Rep cassette insertion at the rbcL locus, combined with nptlII selection for the minichromosome, resulting in the recovery of transgenic tobacco plants (response pg 13).
This is not found persuasive. Applicant has provided no evidence that the citied marker genes are sufficient for transformation of the chloroplasts of the full scope claimed plant and algal species. “The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Given the art at the time of filing expects that extending plastid transformation to other plant species, including those claimed, will require the development of new selectable markers (Bock, pg 222, ¶2-3), evidence for Applicant’s assertions is required.
The application that published as US 20230313212 was filed on 31 August 2021, from a provisional filed less than a year prior; the instant application was filed 20 June 2017 from a foreign priority document filed a year prior. A document with a priority date 4 years after the that of the instant application cannot provide a teaching for the instantly claimed method.
Applicant urges that through minichromosome amplification, they observed strong expression of GFP fused to the gene of interest and demonstrated that fusion with GFP not only facilitated straightforward identification of transgenic plants via confocal microscopy but also significantly enhanced the yield of the GFP-fusion protein compared to the non-fused counterpart (response pg 13).
This is not found persuasive. The instant specification provides no teaching of transformation with a construct encoding GFP fused to the gene of interest or the asserted demonstration or enhanced yield and Applicant has not provided any evidence of this, including over the full scope of claimed plants. The specification provides not teach of how to use reporter genes such as GFP or LacZ in order to select for organellar transformants. “The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Applicant urges that a number of chloroplast-selectable markers are available; the nptll gene serves as a universal marker, the claimed method supports both marker-free and marker-assisted transformations, and GFP fusion has been shown to be a practical and effective strategy for both identifying transgenic plants and increasing protein yield from the transgene cassette. (response pg 13-14).
This is not found persuasive. Applicant has provided no evidence regarding their assertions that the nptll gene serves as a universal marker for plastid transformation or for their assertions regarding use of GFP fusion for both identifying transplastomic plants and increasing protein yield from the transgene cassette.
Applicant has provided no evidence that their method works without use of a selectable marker; the examples in the specification used a selectable marker. Post-filing art teaches selection with a selectable marker is necessary in the early stages of such transformation of tobacco plastids (Jakubiec et al, see, for example, the paragraph spanning the columns on pg 933; the paragraph spanning pg. 934-935; pg 935, right column, ¶2). The specification fails to teach how to transform plastids with the polynucleotide in the absence of a selectable marker.
“The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Applicant urges that the skilled person would know that the organogenesis method is particularly suited for chloroplast transformation, as it enables regeneration of transgenic plants from individual somatic cells through the formation of calli or embryos, and that any tissue culture approach developed for Agrobacterium-mediated transformation using organogenesis from leaves, immature embryos, or meristematic tissue is equally applicable to chloroplast transformation (response pg 4).
This is not found persuasive. Applicant has not provided any support for their assertions, which are mere arguments of counsel. The art teaches that plastid transformation requires specialized tissue culturing and regeneration procedures (Bock, pg 221, ¶6, to pg 219, ¶2 to pg 222, ¶3) and that even the availability of robust tissue culturing and regeneration methods is not enough for achieving plastid transformation is some plant species (Bock; pg 222, ¶2).
The specification does not provide guidance regarding culturing protocols that can be used for the full scope of claimed plants and does not provide teachings regarding modifications to the disclosed methods that could extend its use to the full scope of species encompassed by the claims. Extending methods applicable to the current list of plant species for which plastid transformation works to other plant species, including the full scope of those instantly claimed, would require “optimization work [that] is exceedingly laborious and time consuming and is based largely on the trial-and-error principle” (Bock, pg 222, ¶3).
“The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965).” MPEP 716.01(c) II.
Applicant urges that somatic organogenesis-based plant regeneration protocols are established for many different plant species; the recovery of transgenic plants with transformed chloroplasts is within the standard abilities of the skilled person (response pg 14).
This is not found persuasive. Applicant has not provided any support for their assertions. The art teaches the opposite (see above).
Applicant cites Jiang et al 2025, to assert that there is already a successful example of the presently claimed method being used to transform algae; Applicant has shown that the combination of BCTV Rep and BCTV viral origins worked effectively in soybean, carrot, and even Spirodela and that the MSV and origins of replication functioned successfully in tobacco (response pg 15-16).
This is not found persuasive. Jiang et al 2025, was not sent; thus, it could not be evaluated to see if the methods in Jaing are those of the method as claimed. Applicant has provided no evidence that the BCTV Rep and viral origins worked effectively in soybean, carrot, and Spirodela or that the MSV and origins of replication functioned successfully in tobacco.
Applicant urges that the geminivirus Rep protein has the ability to recruit host DNA replication machinery to the viral origin of replication; the method does not require additional elements beyond geminivirus components to function in the plant species recited in the present claims (response pg 16).
This is not found persuasive. The claims do not require a geminivirus Rep protein. Applicant has provided no evidence to support these mere arguments of counsel.
Applicant urges that plant viruses replicate in the nucleus, and thus the native Rep protein is targeted to the nucleus to support viral replication; it would be clear to the skilled person that, for the system of the presently claimed invention to be functional in plastids, the Rep protein must be specifically targeted to plastids (response pg 16).
This is not found persuasive. The organelle, plant and plant cells of claims 5-7, 9-10 and 17-22 do not have this feature Applicant asserts is required, and the methods of claims 11-12 and 16 also do not have this feature.
Polynucleotide 1(i) has the viral origins of replication outside the flanking sequences. When polynucleotide 1(i) is transformed into plastids, homologous recombination will insert the transgene of interest between the plastid genomic regions delineated by the flanking sequences and the viral origins of replication will be eliminated along with the rest of the vector on which the polynucleotide is located.
The specification does not teach how to transform the plastids of the full scope of plants, it does not teach the full scope of the specialized tissue culturing and regeneration procedures needed for plastid transformation, it does not teach the full scope of species-specific flanking regions, it does not teach DNA delivery methods for the full scope of plants.
Current DNA delivery methods do not work in the full scope of plant species (Maliga, pg 997, left column, ¶1), plastid genomic sequences are not known for the full scope of claimed plants (Wang et al, 2024, Trends in Plant Science, pg 1-16; DOI: 10.1016/j.tplants.2023.12.014; pg 2 ¶2; Figure 1), development of new selectable markers and selection procedures are required for the full scope of plants, and the claimed methods require selectable markers and rep proteins not recited in the claims. Accomplishing these over the full scope of plants will require undue experimentation.
The specification does not teach how to transform the mitochondria with a polynucleotide comprising an organellar transgene cassette comprising an origin of replication derived from a geminivirus, a flanking sequence, a transgene of interest, another flanking sequence, and another origin of replication derived from a geminivirus. Stable mitochondrial transformation via homologous recombination had not been achieved in any plant species, even years after the effective filing date of the instant invention (Wang et al, pg 10, ¶1). Applicant’s teaching in the well-studied tobacco does not make up for the specification’s lack of teaching for the full scope of plants.
Jakubiec et al teaches that extension of organellar transformation with polynucleotide 1(ii) to other organisms will require further development (paragraph spanning pg 937-938). The authors of this reference, which was published 5 years after the filing of the instant priority document, include 3 of the instant inventors.
Applicant is reminded that evidence submitted after final will not be considered unless Applicant provides a showing of good and sufficient reasons of why they were necessary and not earlier presented.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-2, 5-7, and 9-22 are rejected under 35 U.S.C. 103(a) as being unpatentable over Regnard et al (2010, Plant Biotechnol. 8:38-46) in view of Daniell (2008, US 7,354,760) and Selth et al (2002, FEBS Letters 516:179-182).
Due to Applicant’s amendment of the claims, the rejection is modified from the rejection set forth in the Office action mailed 11 March 2025. Applicant’s arguments filed 11 August 2025 have been fully considered but they are not persuasive.
The claims are drawn to a transgene comprising two geminivirus origins of replication flanking construct comprising an organellar promoter, a DNA of interest, and organellar terminator, a method of introducing into the transgene into plants, cells, and organelles and plants, cells, and organelles so produced.
Regnard et al teach Agrobacterium transformation vectors comprising two copies of the BeYDV geminivirus long intergenic region (LIR) and one copy of the BeYDV short intergenic region (SIR) flanking an expression construct encoding a vaccine antigen and the BeYDV rep gene (figure 1; pg 44, right column, ¶2-4) and transient expression in Nicotiana benthamiana (Figures 2-3; pg 44, right column, ¶5, to pg 45, left column, ¶2). The LIR contains the viral origin of replication (pg 39, left column, ¶3). A viral vector is replicationally released from the Agrobacterium vector (paragraph spanning pg 39-40). Regnard et al suggest targeting the vaccine antigen to the chloroplast to increase protein production (pg 44, left column, ¶2).
As the vector two copies of the BeYDV LIR and the LIR contains the viral origin of replication, the vector has two origins of replication, which inherently bind the same Rep protein.
Regnard et al do not teach transformation of the construct into plant plastids.
Daniell teaches plastid transformation vectors encoding anthrax protective antigen F1-V; the vectors comprise an expression cassette comprising a plastid promoter, the aadA selectable marker, the Prrn plastid promoter, the psbA 5’ UTR, a sequence encoding F1-V, the psbA transcriptional terminator, flanked by the tobacco plastid trnI and trnA plastid genes (Figure 13A) and transformation via microprojectile bombardment into tobacco plastids (column 9, lines 5-26; column 10, lines 3-22; examples 1 and 4). The anthrax protective antigen was expressed at high levels in plastids (column 11, line 22, to column 12, line 32).
Daniell also teaches that foreign gene expression has also been achieved in plastids via autonomously replicating vectors (column 5, lines 23-25) and that production of very large amounts of foreign proteins in plastids is possible (column 5, lines 44-53). Daniell also teaches transformation of maize chloroplasts (example 7).
Selth et al teach that the TLCV geminivirus can replicate in bacteria, specifically E. coli and Agrobacterium. Such replication requires an intact rep protein and two copies of the viral ori (paragraph spanning the col