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 .
Status of the Claims
Claims 1, 6-9, and 19-21 are pending.
Claims 1, 6-9, and 19-21 are examined herein.
Claims 1, 6-9, and 19-21 are rejected.
Priority
As stated in previous Action:
Application No. 18/272,978 filed on 07/18/2023 is a PCT of Application No. PCT/JP2022/002162 filed on 01/21/2022 which claims priority to provisional Application No. 63/285/223 filed on 12/02/2021. Application No. 18/272,978 also claims foreign priority to Japanese Application No. JP2021-009001 filed on 01/22/2021.
A certified English translation of the foreign priority document has been provided, dated 01/15/2026.
Upon review, the pending claims dated 01/15/2026 are benefited the following priority dates:
Japanese Application No. JP2021-009001 filed on 01/22/2021 only provides support for claim 20 because the priority document describes the invention comprising a plastid localization signal, and does not describe the method comprising adding a nuclear or mitochondrial localization signal. Therefore, claim 20 is benefitted the priority date of 01/22/2021.
Provisional Application No. 63/285/223 filed on 12/02/2021 only provides support for claim 21 because the priority document only describes the invention comprising a mitochondrial localization signal, and does not describe the method comprising adding a nuclear or plastid localization signal. Therefore, claim 21 is benefitted the priority date of 12/02/2021.
PCT of Application No. PCT/JP2022/002162 filed on 01/21/2022 provides support for all pending claims. Therefore, remaining claims 1, 6-9, and 19 are benefitted the priority date of 01/21/2022.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1, 6-9,19, and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Mok (Mok, B. Y., de Moraes, M. H., Zeng, J., Bosch, D. E., Kotrys, A. V., Raguram, A., ... & Liu, D. R. (2020). A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 583(7817), 631-637) Kang (Kang, B. C., Bae, S. J., Lee, S., Lee, J. S., Kim, A., Lee, H., ... & Kim, J. S. (2021). Chloroplast and mitochondrial DNA editing in plants. Nature Plants, 7(7), 899-905), and Kavipriya (Kavipriya, C., Yuvaraja, A., & Senthil, C. M. (2019). Genetic Transformation Methods for Crop Improvement: A Brief Review. Agricultural Reviews, 40(4).).
This is a maintained rejection from the previous Office Action dated 02/24/2026.
Claim 1 is drawn to a method for editing a plant genomic DNA, comprising converting a target nucleotide on the genomic DNA to another nucleotide, wherein the conversion is carried out with cytidine deaminase which is a protein described in the following (a) or (b):(a) a protein comprising the amino acid sequence as set forth in SEQ ID NO: 35; or (b) a protein comprising an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence as set forth in SEQ ID NO: 35, and having cytidine deaminase activity, wherein an N-terminal portion of the cytidine deaminase and another portion are each fused with a different transcription activator-like effector (TALE), and wherein the conversion comprises introducing a DNA encoding a fusion protein comprising a part of or the entire cytidine deaminase and TALE, to which a nuclear localization signal peptide, a plastid localization signal peptide or a mitochondrial localization signal peptide is added, into a nuclear genome in a plant cell, and then allowing the signal peptide-added fusion protein to express in the plant cell.
Claim 6 is drawn to a plant genome, comprising a plant genomic DNA edited by the method according to claim 1.
Claim 7 is drawn to a plant cell, comprising the plant genome according to claim 6.
Claim 8 is drawn to a seed or a plant, comprising the plant cell according to claim 7.
Claim 9 is drawn to a method for producing a plant having an edited plant genome, the method comprising editing a plant genome by the method for editing a plant genomic DNA according to claim 1.
Claim 19 is drawn to the method according to claim 1, wherein the conversion comprises introducing the DNA encoding the fusion protein comprising the part of or the entire cytidine deaminase and TALE, to which the nuclear localization signal peptide is added, into the nuclear genome in the plant cell.
Claim 21 is drawn to the method according to claim 1, wherein the conversion comprises introducing the DNA encoding the fusion protein comprising the part of or the entire cytidine deaminase and TALE, to which the mitochondrial localization signal peptide is added, into the nuclear genome in the plant cell.
Regarding claim 1, Regarding claim 1, Mok teaches a cytidine deaminase polypeptide sequence (UniProt Accession No. AOA1V2VU04, published online 12/02/2020) that comprises an amino acid sequence with 100% identity to instant SEQ ID NO: 35 (see alignment in Office Action dated 04/29/2025). Mok also teaches the cytidine deaminase (named DddA) is split in half, wherein one half contains the C-terminus and the other half contains the N-terminus (p. 633, section titled Splitting DddAtox into non-toxic halves), and each portion is fused with a different TALE (Fig. 3a). Further, Mok also teaches the cytidine deaminase is used to edit genomes to convert C*G to T*A by expressing two halves of a cytidine deaminase (named DddA) that are fused to two different TALEs and also operably linked to a mitochondrial targeting signal (Fig. 3a and p. 634, section titled Mitochondrial base editing by TALE- DddAtox) in cells.
Regarding claim 19, Mok teaches the TALE-DddAtox can be used for nuclear base editing when a nuclear localization signal is added (p. 634, ¶2).
However, Mok does not explicitly teach:
The method is used for editing plant genomic DNA, and the conversion comprises introducing a DNA encoding a fusion protein comprising the cytidine deaminase and TALE into a nuclear genome in a plant cell, and then allowing the signal peptide-added fusion protein to express in the plant cell (remaining limitations of claim 1).
A plant genome, comprising a plant genomic DNA edited by the method according to claim 1 (claim 6).
A plant cell, comprising the plant genome according to claim 6 (claim 7).
A seed or a plant, comprising the plant cell according to claim 7 (claim 8).
The method according to claim 1, wherein the conversion comprises introducing the DNA encoding the fusion protein comprising the part of or the entire cytidine deaminase and TALE, to which the nuclear localization signal peptide is added, into the nuclear genome in the plant cell (claim 19).
The method according to claim 1, wherein the conversion comprises introducing the DNA encoding the fusion protein comprising the part of or the entire cytidine deaminase and TALE, to which the mitochondrial localization signal peptide is added, into the nuclear genome in the plant cell (claim 21).
Regarding the remaining limitations of claim 1 and claim 19, in analogous art Kang teaches applying the method described by Mok to lettuce and rapeseed plant cells (abstract, p. 899, ¶2) to edit mitochondrial and chloroplast DNA by adding a chloroplast transit peptide (CTP) or a mitochondrial targeting sequence (MTS) respectively to generate fusion peptides (abstract, p. 899, ¶3). The fusion peptides are expressed via transient expression of plasmids and via DNA-free in vitro transcribed ddCBE mRNA using polyethylene glycol (PEG)-mediated transfection methods. In other analogous art, Kavipriya teaches a review of various transformation methods (title). In this review, Kavipriya teaches alternative methods of plant cell transformation including, e.g., agrobacterium-mediated transformation which generates high transformation frequencies and stable transformants (i.e. the plasmids are introduced into the nuclear genomes) (Table 2).
Regarding claim 6, Kang teaches mitochondria and chloroplasts contain their own genomes, and the DddA-derived cytosine deaminase base editor induced base editing in lettuce or rapeseed calli at frequencies of up to 25% in mitochondria (abstract) and 38% in chloroplasts (i.e. therefore Kang teaches a plant genome comprising edited plant genomic DNA).
Regarding claim 7, Kang teaches rapeseed and lettuce protoplasts with base editing in in chloroplasts at frequencies of 15% and 30% respectively (p. 899, ¶4), and base editing in mitochondria at frequencies of 11% and 23% respectively (p. 900, ¶2) (i.e. therefore Kang teaches a plant cell comprising the edited plant genome).
Regarding claims 8-9, Kang teaches regenerating DNA-edited callus from the protoplasts, and further regenerating DNA-edited plants from the callus (abstract, Fig. 3f) (i.e. a plant comprising the plant cell, and also a method of producing a plant having the edited plant genome).
Regarding claim 21, Kang teaches the localization signal fused to the base-editing peptide used for base editing in the plant cells is a mitochondrial targeting sequence (p. 899, ¶3) (i.e. a mitochondrial localization signal peptide).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention taught by Mok to include the limitations of Kang and Kavipriya to arrive at the instantly claimed method with a reasonable expectation of success because Kang explicitly teaches the DddAtox cytidine deaminase-TALE complex taught by Mok can also be used to edit mitochondrial genomes of plant cells (title, abstract), and Kavipriya teaches various transient and stable transformation methods are available as functional alternatives. One having ordinary skill in the art would have been motivated to combine the teachings because Kang teaches successfully editing mitochondrial DNA in plants using the DddAtox-TALE base editor of Mok, and Kavipriya teaches PEG-induced DNA uptake (i.e. whether transient transfection or stable transformation) uses protoplasts which are often difficult to regenerate plants from (p. 286, ¶1, Table 2), a demerit not associated with other methods including the known, functionally equivalent methods for expressing plasmid DNA including stable transformation via agrobacterium-mediated transformation. Furthermore, it would have been obvious to apply the method of adding a nuclear localization signal for nuclear genome base editing as taught by Mok to plants and plant cells as taught by Kang for the same purpose that is targeted base editing of the nuclear genome in plants rather than mammalian cells.
Claims 1 and 20 are separately rejected under 35 U.S.C. 103 as being unpatentable over Mok (Mok, B. Y., de Moraes, M. H., Zeng, J., Bosch, D. E., Kotrys, A. V., Raguram, A., ... & Liu, D. R. (2020). A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 583(7817), 631-637) and Zhang (US-20200181623-A1).
This is a maintained rejection from the previous Office Action dated 02/24/2026.
Claim 1 is drawn to a method for editing a plant genomic DNA, comprising converting a target nucleotide on the genomic DNA to another nucleotide, wherein the conversion is carried out with cytidine deaminase which is a protein described in the following (a) or (b):(a) a protein comprising the amino acid sequence as set forth in SEQ ID NO: 35; or (b) a protein comprising an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence as set forth in SEQ ID NO: 35, and having cytidine deaminase activity, wherein an N-terminal portion of the cytidine deaminase and another portion are each fused with a different transcription activator-like effector (TALE), and wherein the conversion comprises introducing a DNA encoding a fusion protein comprising a part of or the entire cytidine deaminase and TALE, to which a nuclear localization signal peptide, a plastid localization signal peptide or a mitochondrial localization signal peptide is added, into a nuclear genome in a plant cell, and then allowing the signal peptide-added fusion protein to express in the plant cell.
Claim 20 is drawn to the method according to claim 1, wherein the conversion comprises introducing the DNA encoding the fusion protein comprising the part of or the entire cytidine deaminase and TALE, to which the plastid localization signal peptide is added, into the nuclear genome in the plant cell.
Regarding claim 1, Regarding claim 1, Mok teaches a cytidine deaminase polypeptide sequence (UniProt Accession No. AOA1V2VU04, published online 12/02/2020) that comprises an amino acid sequence with 100% identity to instant SEQ ID NO: 35 (see alignment in Office Action dated 04/29/2025). Mok also teaches the cytidine deaminase (named DddA) is split in half, wherein one half contains the C-terminus and the other half contains the N-terminus (p. 633, section titled Splitting DddAtox into non-toxic halves), and each portion is fused with a different TALE (Fig. 3a). Further, Mok also teaches the cytidine deaminase is used to edit genomes to convert C*G to T*A by expressing two halves of a cytidine deaminase (named DddA) that are fused to two different TALEs and also operably linked to a mitochondrial targeting signal (Fig. 3a and p. 634, section titled Mitochondrial base editing by TALE- DddAtox) in cells.
However, Mok does not explicitly teach:
The method is used for editing plant genomic DNA, and the conversion comprises introducing a DNA encoding a fusion protein comprising the cytidine deaminase and TALE into a nuclear genome in a plant cell, and then allowing the signal peptide-added fusion protein to express in the plant cell (remaining limitations of claim 1).
wherein the conversion comprises introducing the DNA encoding the fusion protein comprising the part of or the entire cytidine deaminase and TALE, to which the plastid localization signal peptide is added, into the nuclear genome in the plant cell (claim 20).
Regarding the remaining limitations of claim 1, in analogous art Zhang teaches a similar method of modifying a target locus of interest within a plant cell using cytidine deaminase, wherein the method modifies the cell by manipulation of one or more target sequences at genomic loci of interest (claims 1, 24, and 26 of Zhang), and the modified cell comprises a uracil or thymine in replacement of a cytosine in the target locus (claim 41 of Zhang). Zhang teaches in particular embodiments, the cytosine deaminase functionalized crispr system is introduced for stable integration into the genome of a plant cell, and the design of the transformation vector or the expression system can be adjusted depending on for when, where and under what conditions the guide RNA and/or fusion protein of cytidine deaminase and Cpf1 are expressed (¶0524). Zhang further teaches the fusion protein comprises a nuclear localization signal (claim 18 of Zhang).
Regarding claim 20, Zhang teaches alternatively, it is envisaged to target one or more of the CD-functionalized CRISPR components to the plant chloroplast by incorporating in the expression construct a sequence encoding a chloroplast transit peptide (CTP) or plastid transit peptide, operably linked to the 5′ region of the sequence encoding the fusion protein of cytidine deaminase and Cpf1 (¶0542).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention taught by Mok to include the limitations of Zhang to arrive at the instantly claimed method with a reasonable expectation of success because both inventions are directed to targeted base editing in cells using cytidine deaminases, and one of ordinary skill could apply the DddAtox-TALE cytidine deaminase of Mok to the method of editing chloroplast DNA of plant cells by incorporating a plastid localization signal as taught by Zhang without encountering any special technical difficulties. One having ordinary skill in the art would have been motivated to do so because it would be obvious to use one known cytidine deaminase base editor rather than another for the same purpose that is targeted nucleic acid editing (title) including targeting to the chloroplast as taught by Zhang (¶0542).
Response to Arguments
Applicant argues beginning on p. 2 of remarks dated 05/12/2026 the
following arguments:
Applicant first argues that one having ordinary skill in the art would not have had a reason to modify Mok in the manner proposed in the first place. Mok clearly discloses gene editing performed via transient expression. On the other hand, the claimed methods require gene editing performed by stable expression. However, it would have been thought that stable expression would give rise to undesirable transgenics, which would violate the Cartagena Protocol. As such, the skilled artisan would not even attempt to modify Mok so as to use stable expression instead of transient expression. Furthermore, it would not have been expected that the claimed method yields T-DNA transgene-free plants (null segregants) in T2 plants. See, for example, paragraphs [0074] and [0075] of the specification as filed.
Beginning on p. 2 of the declaration dated 05/12/2026, the declaration states:
Why one skilled in the art would not want to modify Mok to stably express CD-TALE
First, I understand that there is a question about whether the skilled artisan would have wanted to modify Mok so as to stably express CD-TALE. According to my experience and knowledge, I believe that a skilled artisan would not seek to modify Mok so as to stably express CD-TALE, at least because they would wish to avoid transgenics. In this technical field, due to the existence of the Cartagena Protocol on Biosafety¹, also known as the "Cartagena Act," there is a prevailing view that it is better to avoid genetic modification of plants as much as possible. See paragraph [0004] of the specification as filed. Therefore, methods aiming for null- segregants through transient expression are being chosen in the art.
On the other hand, the claimed method yields T-DNA transgene-free plants (null segregants) in T2 plants. See, for example, paragraphs [0074] and [0075] of the specification as filed. As such, the claimed method is very beneficial at least in that it does not violate the regulations of the Cartagena Protocol. The Cartagena Protocol varies from country to country, so it is difficult to make a general statement, but at least in the United States and Japan, plant breeding can be carried out using the claimed method without violating the Cartagena Protocol, due to the lack of transgenics.
Examiner’s response:
This argument has been fully considered and is not persuasive because the method of producing stable transgenics expressing gene-editing machinery followed by selfing the transgenic plant (as is described in the instant spec. and argued here) is a common method utilized in the art to obtain transgene-free edited plants (See Khatodia, p. 6, section titled CRISPR Achievements in Plants, ¶1; and Figure 5). This commonly used method is not novel and would be obvious to do to remove transgenes from gene-edited plants and avoid violating regulations such as The Cartagena Protocol described by Applicant. Additionally, stably transformed plants with transgenes/ T-DNA make up the vast majority of crops grown in the United States (See Dodson, first figure). Therefore, while in some instances, organizations, and/ or countries it may be desirable to avoid transgenes in crops, clearly there is still use of transgenes/ T-DNA in crops in at least the US, and it would be obvious to use stable expression as an alternative to transient expression to produce the gene-edited plants. Thus, the argument that the skilled artisan would not even attempt to modify Mok so as to use stable expression instead of transient expression is not found persuasive.
Applicant argues beginning on p. 4 of remarks dated 05/12/2026 the
following arguments:
Next, Applicant argues the Declaration explains that homoplasmic editing is only known to be obtained through stable expression, not transient expression. As disclosed in at least paragraphs [0070] and [0082] and Figure 1b in the specification as filed, homoplasmic editing is obtained through stable expression. Such homoplasmic editing was not known to be possible using transient expression.
Beginning on p. 3 of the declaration dated 05/12/2026, the declaration states:
7. Homoplasmic editing is only known to be obtained through stable expression, not
transient expression.
Next, I understand that there is a question about whether homoplasmic editing could have been achieved using transient expression. At least to date, there have been no reports of homoplasmic single-nucleotide editing being performed by transient expression of TALE-CD in plant genome editing. Therefore, it is believed that stable expression must be employed in order to obtain homoplasmic editing.
In the present invention, because the intracellular presence time of TALE-CD is significantly longer with stable expression compared to transient expression, homoplasmic single-nucleotide editing is possible. For example, disclosed embodiments used the RPS5A promoter, which is strongly expressed in stem cells (egg cells and germline cells). See at least paragraphs [0070] and [0082] and Figure 1b in the specification as filed. Once homoplasmic editing occurs within a stem cell, all tissues that divide and differentiate from that stem cell will be composed of cells that have undergone homoplasmic editing. As a result, homoplasmic editing can be performed throughout the entire plant. It is my belief and understanding that such homoplasmic editing cannot occur when transient expression is employed.
Examiner’s response:
This argument has been fully considered and is not persuasive because Kang teaches transient expression of cp-DdCBE targeted to the 16S rRNA gene in lettuce protoplasts, followed by regeneration of calli and plantlets on medium comprising streptomycin or spectinomycin produced calli and plants with C to T editing frequencies up to 99% at the targeted position (abstract, Fig. 3a and p. 903, ¶1). Thus, Kang teaches near homoplasmic editing can occur when transient expression is employed. One of ordinary skill in the art would reasonably expect that, as argued above, because the intracellular presence time of TALE-CD could be significantly longer with stable transformation compared to transient expression, the stable integration and continuous expression of the cytidine deaminase would be able to achieve editing of the remaining 1%, and thus achieve homoplasmic editing.
Applicant argues beginning on p. 4 of remarks dated 05/12/2026 the
following arguments:
Next, Applicant argues the Declaration explains the benefits of the claimed embodiments. In addition to avoiding violating the Cartagena Protocol, the claimed methods, which achieve 100% editing efficiency, allow for practical improvements over prior art methods, which achieve only about 40% editing efficiency. For example, using the claimed embodiments, it is possible to obtain mutants which exhibit phenotypes that cannot be further screened using indicators such as drug resistance.
Beginning on p. 4 of the declaration dated 05/12/2026, the declaration states:
8. The practical benefits of homoplasmic editing
Next, I understand that it would be helpful to describe the practical benefits and significance of homoplasmic editing. I believe that there is a very significant difference in editing efficiency between an editing frequency of 100% achieved in the embodiments of the present application and an editing frequency of only about 40% achieved in the prior art. The nearly 100% editing efficiency is disclosed in the specification as follows:
Plastid genome: Paragraphs [0071]-[0073], and Figures 4a-4g. The number in the "homo" box is the number of individuals which have a 100% editing efficiency.
Mitochondrial genome: Paragraphs [0084]-[0085] and Figures 10b and 10d
Nuclear genome: Paragraphs [0097]-[0098] and Figures 25 and 26.
With an editing efficiency of about 40%, the plant (plant body) that retains the mutation is merely a chimera, and the possibility of obtaining a homoplasmic edited plant with the target base on the genome completely edited is considered to be zero. Therefore, obtaining a desired complete mutant (not a chimera) is extremely difficult with the transient expression of TALE- CD disclosed by Kang and Mok. In particular, to obtain the desired edited plants, for example, a streptomycin-resistant plant or a spectinomycin-resistant plant, using the technology of Kang or Mok, it is necessary to further screen using indicators such as drug resistance. See abstract of Kang. This is time-consuming and difficult. On the other hand, the method of the disclosed embodiments makes it possible to obtain the desired mutant (homoplasmic edited plant) without further screening. See paragraph [0075] of the specification as filed, for example.
Furthermore, when the goal is to obtain mutants exhibiting phenotypes that cannot be further screened using indicators such as drug resistance, for example, if the aim is to obtain plants with improved growth retardation by replacing 1178C of atp1, which is one of the causes of growth retardation in otp87 mutants (plants with mutations in the mitochondrial genome), with T, it is extremely difficult to obtain the desired edited plants unless homoplasmic editing is possible at the genome editing stage. See paragraph [0090] of the specification as filed.
This is because there is no simple screening method using drugs or other means for plants with improved growth retardation. As shown in the present paragraph [0090], plants with improved growth retardation can be obtained because 1178C can be homoplasmically replaced with T by the method of the claimed embodiments. However, it is extremely difficult to obtain such plants with improved growth retardation using genome editing technologies such as those of Kang and Mok.
Thus, compared to prior art which can only achieve an editing efficiency of about 40%, the claimed embodiments are practically much improved due to the editing efficiency which can be achieved through homoplasmic expression. I believe this to be an unexpected and remarkable effect.
Examiner’s response:
This is not found persuasive. With regard to Applicant’s argument that Applicant has offered evidence of unexpected and unobvious results, pursuant to MPEP 716.02(b), the evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992) (Mere conclusions in appellants’ brief that the claimed polymer had an unexpectedly increased impact strength "are not entitled to the weight of conclusions accompanying the evidence, either in the specification or in a declaration."); Ex parte C, 27 USPQ2d 1492 (Bd. Pat. App. & Inter. 1992) (Applicant alleged unexpected results with regard to the claimed soybean plant, however there was no basis for judging the practical significance of data with regard to maturity date, flowering date, flower color, or height of the plant.). In the instant case, Applicant alleges the unexpected result of improved editing efficiency and homoplasmic editing via stable expression of the TALE-CDs. This does not appear unexpected and unobvious because Kang teaches transient expression of the TALE-CDs targeted the 16S rRNA gene in the plastid genome, in combination with regeneration on spectinomycin, could actually achieve 99% editing efficiency and calli and regenerated plantlets (Fig. 3e). One of ordinary skill in the art would reasonably expect that longer expression time via, e.g. stable expression, could achieve the remainder 1% editing. Thus, it does not appear unexpected or unobvious that stable expression could achieve homoplasmic editing.
It is also noted that Applicant states “nearly 100% editing” in this argument of the declaration. Applicant states in the disclosure:
“The present inventors have found that, by using the above-described method for editing a plant genome according to the present invention, the target C:G pairs contained in the plant genome (nuclear genome, plastid genome, and mitochondrial genome) can be homoplasmically modified, namely, if taking the plastid genome as an example, almost all of the target C:G pairs in about 1000 copies or more of plastid genomes contained in a cell in the plant can be converted to T:A pairs” (¶0012).
“the target nucleotide C in a nuclear genomic DNA, a plastid genomic DNA and a mitochondrial genomic DNA can be converted to T, homoplasmically (a state in which the same mutations are kept in all of cells and tissues, or in plants)…” (¶0051).
“These results suggest that the target nucleotide of T1 transformed with the ptpTALECD expression vector be homoplasmically substituted at a high frequency, and that the mutation be stably maintained throughout the growth process” (¶0072).
Applicant also states on p. 6 of remarks “However, homoplasmic editing (almost 100% editing rate) was achieved by stably expressing TALECD”.
While the term homoplasmic/ homoplasmically is understood to mean having 100%, uniform editing of targeted nucleotides in chloroplast, mitochondria, or nuclear DNA, there disclosure presents possible alternative meanings as to what is considered “homoplasmic editing”. Especially in the case of “nearly 100% editing efficiency” as stated in the declaration, the results do not appear unexpected and unobvious because Kang teaches up to achieve 99% editing efficiency (i.e. nearly 100%editing efficiency as stated in declaration) and calli and regenerated plantlets (Fig. 3e), and one of ordinary skill would expect that prolonged expression of the TALE-CDs could achieve at least the same editing efficiency as transient expression.
Further, even if Applicant can make such a showing, MPEP 716.02(c) provides that the evidence of unexpected results must be weighed against evidence supporting prima facie obviousness in making a final determination of the obviousness of the claimed invention. MPEP 716.02(c) directs the examiner to MPEP 716.01(d), which establishes that although the record may establish evidence of secondary considerations which are indicia of nonobviousness, the record may also establish such a strong case of obviousness that the objective evidence of nonobviousness is not sufficient to outweigh the evidence of obviousness. Newell Cos. v. Kenney Mfg. Co., 864 F.2d 757, 769, 9 USPQ2d 1417, 1427 (Fed. Cir. 1988), cert. denied, 493 U.S. 814 (1989); Richardson-Vicks, Inc., v. The Upjohn Co., 122 F.3d 1476, 1484, 44 USPQ2d 1181, 1187 (Fed. Cir. 1997) (showing of unexpected results and commercial success of claimed ibuprofen and pseudoephedrine combination in single tablet form, while supported by substantial evidence, held not to overcome strong prima facie case of obviousness). The showing, when made, must outweigh the rationale in support of a finding of prima facie obviousness provided in the 103 rejection(s). Here, the prior art is strong and Kang teaches using the method of Mok to edit plastid genomes of plants via transient expression of TALE-CDs. The missing teaching by Kang is that of stable transformation and expression of the TALE-CDs in plants. Because transient and stable transformation are functional alternatives as taught by Kavipriya, and one of ordinary skill in the art would understand that increasing expression time would be expected to increase editing frequency, it would be obvious to perform the method of stably expressing the TALE-CDs in plants. In view of the foregoing, Applicant’s evidence is not deemed to outweigh the basis for the rejection.
Finally, MPEP 716.02(d) provides that whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980). In this case, the scope of the claims appears commensurate with the evidence.
Applicant argues beginning on p. 4 of remarks dated 05/12/2026 the
following arguments:
Applicant argues the Declaration provides comment regarding off-target mutations, and explains that, based on Kang, if stable expression was used, the off-target mutation rate would have been expected to be several tens or more compared to the transient expression of Kang. Thus, the actual observed single-digit off-target mutation rate is unexpectedly good.
Beginning on p. 5 of the declaration dated 05/12/2026, the declaration states:
9. Off-Target Mutations
Next, I wish to comment on off-target mutations. Kang discloses a 1.2% to 4.1% off- target mutation rate in the case of transient expression. In particular, Kang discloses the following:
We also analysed the off-target activity of the TALE deaminase targeted to the 16S
rRNA site in protoplasts, calli and shoots. No off-target mutations were detectably
induced in antibiotic-resistant calli or shoots, which were derived from single cells,
in the vicinity (±50 base pairs) of the target site (Supplementary Fig. 9) or at the
top five candidate off-target sites in the chloroplast genome, which were chosen on
the basis of sequence homology (Supplementary Fig. 10). In contrast, when
plasmids encoding the DdCBE pair were used to transfect protoplasts, off-target
TC-to-TT mutations were induced in the proximity of the target site and at three of
the five candidate off-target sites with low frequencies that ranged from 1.2% to
4.1% (Supplementary Fig. 10). The use of in vitro transcripts (mRNA) instead of
plasmids encoding the TALE deaminase largely avoided these off-target activities
in protoplasts (Fig. 4). These results suggest that overexpression or prolonged,
plasmid-based expression of DdCBEs can give rise to off-target mutations and that
transient, mRNA-based expression using mRNA is desirable for avoiding off-target
base editing.
See also Figure 3a of Kang. However, even if the expression of Kang leading to an off-target mutation rate of 1.2 to 4.1% is transient expression, if stable expression was used, the off-target mutation rate would be higher.
In the specification of the present application, low off-target mutations are discussed in paragraphs [0073], [0087], and [0099]. Regarding the mitochondrial genome, paragraph [0087] states that "the frequency of off-target mutations outside the target widow has been shown to be 10% or less in various plants." Off-target mutations in the nuclear genome has not been investigated.
Regarding off-target mutations in the plastid genome, Nakazato et al., (see attached) investigated eight T1 generations targeting 16S rRNA (Fig. 2a), three T2 generations (Fig. 2c), five T1 generations targeting rpoC1 (Fig. 2d), and three T1 generations targeting psbA (Fig. 2e). Of these, only two T1 generations targeting 16S rRNA showed off-target mutations at a frequency of 10% or more. Therefore, the frequency of off-target mutations was less than 10% in most individuals if stable expression was used.
Meanwhile, referring to the graph at the bottom of Fig. 4 in Kang, it is shown that the off- target mutation rate for G911, G921, and G941 is 1%-2% in the case of transient expression by plasmid. On the other hand, in the case of expression by mRNA, it is 0.1%-0.5%, which is about an order of magnitude lower than in the case of transient expression by plasmid. The off-target mutation rate of transient expression of TALE-CD by plasmid is about 4-15 times higher (about 15 times higher for G911) than that of transient expression of TALE-CD by mRNA. Considering this point, it is likely that the off-target mutation rate could be several tens or more in the case of stable expression, which expresses TALE-CD for an even longer period than transient expression by plasmid. In view of this, I consider the fact that the off-target mutation rate in the claimed embodiments is at most a few percent to be an unexpected result.
Examiner’s response:
This is not found persuasive. With regard to Applicant’s argument that Applicant has offered evidence of unexpected and unobvious results, pursuant to MPEP 716.02(b), the evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992) (Mere conclusions in appellants’ brief that the claimed polymer had an unexpectedly increased impact strength "are not entitled to the weight of conclusions accompanying the evidence, either in the specification or in a declaration."); Ex parte C, 27 USPQ2d 1492 (Bd. Pat. App. & Inter. 1992) (Applicant alleged unexpected results with regard to the claimed soybean plant, however there was no basis for judging the practical significance of data with regard to maturity date, flowering date, flower color, or height of the plant.). In the instant case, Applicant alleges the unexpected result of low off-target mutation rate. Applicant evidences 1 of 14 plants with edited plastids had off target mutations, and the remainder had no off-target mutations (¶73 and p. 27, table 2). Applicant also evidences off-target mutation frequency up to 10% in edited mitochondria genomes (¶87). Applicant also provides other data on off-target mutations (Fig. 27), with frequency of plants having off-target mutations from 0.03% to 50%. Variation in off-target mutations is to be expected in generating gene-edited plants and these frequencies do not appear unexpectedly low and are therefore not unexpected or unobvious.
Further, even if Applicant can make such a showing, MPEP 716.02(c) provides that the evidence of unexpected results must be weighed against evidence supporting prima facie obviousness in making a final determination of the obviousness of the claimed invention. MPEP 716.02(c) directs the examiner to MPEP 716.01(d), which establishes that although the record may establish evidence of secondary considerations which are indicia of nonobviousness, the record may also establish such a strong case of obviousness that the objective evidence of nonobviousness is not sufficient to outweigh the evidence of obviousness. Newell Cos. v. Kenney Mfg. Co., 864 F.2d 757, 769, 9 USPQ2d 1417, 1427 (Fed. Cir. 1988), cert. denied, 493 U.S. 814 (1989); Richardson-Vicks, Inc., v. The Upjohn Co., 122 F.3d 1476, 1484, 44 USPQ2d 1181, 1187 (Fed. Cir. 1997) (showing of unexpected results and commercial success of claimed ibuprofen and pseudoephedrine combination in single tablet form, while supported by substantial evidence, held not to overcome strong prima facie case of obviousness). The showing, when made, must outweigh the rationale in support of a finding of prima facie obviousness provided in the 103 rejection(s). Here, the prior art is strong and Kang teaches using the method of Mok to edit plastid genomes of plants via transient expression of TALE-CDs. The missing teaching is that of stable transformation and expression of the TALE-CDs in plants. Because transient and stable transformation are functional alternatives, and one of ordinary skill in the art would understand that increasing expression time would be expected to increase editing frequency, it would be obvious to perform the method of stably expressing the TALE-CDs in plants. In view of the foregoing, Applicant’s evidence is not deemed to outweigh the basis for the rejection.
Finally, MPEP 716.02(d) provides that whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980). In this case, the scope of the claims do not appear commensurate with the evidence. This is because the disclosure evidences high variability in off-target mutations using the claimed invention. Because the instantly claimed invention alone does not consistently produce the alleged unexpected result of low off-target mutations, some other factors or conditions beside stable vs transient transformation are responsible for the alleged low off-target mutations that do not appear in the claims.
Applicant argues beginning on p. 4 of remarks dated 05/12/2026 the
following arguments:
Next, Applicant comments on the data of the specification. In particular, it is noted that even heteroplasmic editing is significantly more efficient than the editing efficiency of Kang and Mok. Regarding chloroplast genome targets, heteroplasmy or chimera (h/c) was obtained in up to 67% of T1 plants (Target rpoCl, 1397N-1397C, 3rd G) depending on the construct and target sequence (Fig. 4a-c). Regarding mitochondrial genome targets, h/c mutations were obtained in up to 67% of T1 plants (1397N-1397C, 7th G) depending on the construct (Fig. 10b).
Even in cases where genome editing was present only partially, indicating heteroplasmy or chimerism, homoplasmic lines can be obtained in the next generation. For example, in the T2 plants, all 10 (Figure 7a (16S rRNA 1397CN 2)) plants and all 8 plants (Figure 7a (16S rRNA 1397CN 8)) examined showed homoplasmic editing at the 5th G position.
Additionally, Figure 4g shows that for T1 plants, in both 11DAS and 23DAS, T1 plants with 100% editing efficiency of target base (homoplasmic editing) were 42% (almost half). This demonstrates as superior efficiency as compared to the cited art. In Kang (plant) and Mok (animal), T1 plants with 100% editing efficiency of target base were not obtained at all, i.e. 0%. Figure 10d shows similar subject matter.
Examiner’s response:
Applicant essentially argues superior editing efficiency as compared to the cited art. This is not persuasive because Kang does teach achieving 100% homoplasmic editing of the 16S rRNA gene in calli and T0 plantlets regenerated on media supplemented with spectinomycin (abstract, Fig. 3a). It would also be expected that prolonged expression of the editing machinery, e.g. by stable expression, would greatly increase editing efficiency. Thus, the results do not appear superior in view of what is known in the prior art. Additionally, it is further noted it would still be obvious to combine the teachings to arrive at the instant method and plants produced from the method, any by doing so one of ordinary skill in the art would observe the inherent results of increased editing frequency.
Applicant argues beginning on p. 5 of remarks dated 05/12/2026 the
following arguments:
Finally, Applicant notes that the Office Action also refers to Kavipriya et al. to support the idea of that "various transient and stable transformation methods are available as functional alternatives." See page 7, lines 15-20 and page 8, lines 14-23 of the pending Office Action. However, Applicant respectfully disagrees with the Examiner's comment that Kavipriya (Table 2 in particular) discloses that transient and stable transformation methods are "functional alternatives." Rather, Table 2 provides a discussion of various gene transformation methods, with the merits and demerits of each provided. There is no comment in Kavipriya which states that transient and stable transformation methods are "functional alternatives."
Examiner’s response:
This argument has been fully considered and is not persuasive because the Kavipriya document provides a brief review of genetic transformation methods for crop improvement (title) and provides a list of the various gene delivery methods that are known in the art and that all achieve plant transformation (Table 1). Thus, Kavipriya does provide various transient and stable transformation methods are available as functional alternatives despite Applicant’s argument.
Applicant argues beginning on p. 6 of remarks dated 05/12/2026 the
following arguments:
Claims 1 and 20 are separately rejected under 35 U.S.C. 103 as being unpatentable over Mok and Zhang (U.S. Patent Application Publication No. 2020/0181623 Al).
The Examiner argues that Mok discloses the embodiments as claimed, with the exception of teaching (i) that the method is used for editing plant genomic DNA, and the conversion comprises introducing a DNA encoding a fusion protein comprising the cytidine deaminase and TALE into a nuclear genome in a plant cell, and then allowing the signal peptide-added fusion protein to express in the plant cell, and (ii) that the conversion comprises introducing the DNA encoding the fusion protein comprising the part of or the entire cytidine deaminase and TALE, to which the plastid localization signal peptide is added, into the nuclear genome in the plant cell. The Examiner relies on Zhang to provide this teaching.
Firstly, Applicant respectfully submits that claims 1 and 20 are patentable over the combination of Mok and Zhang for similar reasons as discussed above with respect to the combination of Mok and Kang.
Additionally, in the claimed embodiments, by stably expressing TALECD, the plastid genome, mitochondrial genome and nuclear genome are homoplasmically edited. See for example paragraphs [0012], [0072], [0085], etc. of the specification. The editing frequency of animal mitochondrial genomes by the method of Mok is low, with an average editing efficiency of 43% at three sites even when using the most efficient G1333-split DdCBE. See page 636, left column, lines 18-24 of Mok.
However, upon reviewing Mok, a person skilled in the art would assume that this editing efficiency is equivalent in plants. Thus, they would have thought that homoplasmic editing would be difficult if the method of Mok was directly applied to the mitochondrial or plastid genome of plants. However, homoplasmic editing (almost 100% editing rate) was achieved by stably expressing TALECD. This is an unexpected effect.
Examiner’s response:
This is not found persuasive. With regard to Applicant’s argument that Applicant has offered evidence of unexpected and unobvious results, pursuant to MPEP 716.02(b), the evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992) (Mere conclusions in appellants’ brief that the claimed polymer had an unexpectedly increased impact strength "are not entitled to the weight of conclusions accompanying the evidence, either in the specification or in a declaration."); Ex parte C, 27 USPQ2d 1492 (Bd. Pat. App. & Inter. 1992) (Applicant alleged unexpected results with regard to the claimed soybean plant, however there was no basis for judging the practical significance of data with regard to maturity date, flowering date, flower color, or height of the plant.). In the instant case, Applicant alleges the unexpected result of homoplasmic editing of plastids by stably expressing TALECD in plant cells (limitations claim 20 is drawn to). This result does appear unexpected, as the different between 43% and nearly 100% is a large difference. However, the evidence does not appear to be of practical significance because various factors affect expression and editing efficiency, for example at least the TALE-CD target and design, the cell species that is being edited, and the construct elements and design (promoter, enhancer, terminator, choice of codon usage, etc.).
Further, even if Applicant can make such a showing, MPEP 716.02(c) provides that the evidence of unexpected results must be weighed against evidence supporting prima facie obviousness in making a final determination of the obviousness of the claimed invention. MPEP 716.02(c) directs the examiner to MPEP 716.01(d), which establishes that although the record may establish evidence of secondary considerations which are indicia of nonobviousness, the record may also establish such a strong case of obviousness that the objective evidence of nonobviousness is not sufficient to outweigh the evidence of obviousness. Newell Cos. v. Kenney Mfg. Co., 864 F.2d 757, 769, 9 USPQ2d 1417, 1427 (Fed. Cir. 1988), cert. denied, 493 U.S. 814 (1989); Richardson-Vicks, Inc., v. The Upjohn Co., 122 F.3d 1476, 1484, 44 USPQ2d 1181, 1187 (Fed. Cir. 1997) (showing of unexpected results and commercial success of claimed ibuprofen and pseudoephedrine combination in single tablet form, while supported by substantial evidence, held not to overcome strong prima facie case of obviousness). The showing, when made, must outweigh the rationale in support of a finding of prima facie obviousness provided in the 103 rejection(s). Here, the prior art is strong and an obviousness rejection can be made with two references. Mok teaches the majority of the limitations, and Zhang compensates for the limitations of introducing the cytidine deaminase fusion protein which comprises a plastid localization signal peptide into in plant genomic DNA to edit plant genomic DNA. There is also strong motivation to combine the teachings and a reasonable expectation of success. In view of the foregoing, Applicant’s evidence is not deemed to outweigh the basis for the rejection.
Finally, MPEP 716.02(d) provides that whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980). In this case, the scope of the claims appears commensurate with the evidence.
Conclusion and Inquiries
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA N STOCKDALE whose telephone number is (703)756-5395. The examiner can normally be reached M-F 8:30-5:00 CT.
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JESSICA N. STOCKDALE
Examiner
Art Unit 1663
/JESSICA NICOLE STOCKDALE/ Examiner, Art Unit 1663
/CHARLES LOGSDON/ Primary Examiner, Art Unit 1662
Prosecution Insights