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 .
Election of Species
Applicant's election without traverse of SEQ ID NO: 20 and the substitution mutation of the elected sequence of the amino acid threonine at position 139 (claims 21xxii and 22xxii) in the reply filed on 07/06/2026 is acknowledged.
Status of Claims
Claims 17-36 are pending and are examined in this Office action.
Priority
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in parent Application No. 15/539,065, filed on 06/22/2017.
Claim Objections
Claim 17 is objected to for the wording in line 4, “alters the amino acid sequence of the CENH3 protein in an α2-helix”. It sounds as though the CENH3 protein is encompassed by an α2-helix. For clarity, it is suggested to amend the phrase to --- alters the amino acid sequence of the CENH3 protein in an α2-helix domain of the CENH3 protein---.
Claim 22 is objected to because of the following informalities: claim 22i-xxii recites the phrase “is substituted for”. For clarity, it is suggested to amend the phrase to ---is substituted with---.
Appropriate correction is required.
Claim Interpretation
Claim 17 recites “wherein the nucleotide sequence comprises at least one introduced mutation that alters the amino acid sequence of the CENH3 protein in an α2-helix”. Applicant has elected SEQ ID NO: 20, the amino acid sequence of the wildtype Zea mays CENH3, and the substitution of the amino acid threonine at position 139 of SEQ ID NO: 20. The instant Specification describes the α2-helix of Zea mays CENH3 as corresponding to the amino acid sequence from position 107 to position 135 as set forth in SEQ ID NO: 20 (page 8, lines 22-23). Position 139 of SEQ ID NO: 20 is in the loop2 region, which corresponds to the amino acid sequence from position 136-142 of SEQ ID NO: 20 (page 9, line 5). Therefore, claims 21 and 22 will be interpreted as comprising at least one introduced mutation that alters the amino acid sequence of the CENH3 protein in an α2-helix (according to claim 17), and the elected amino acid substitution at position 139 of SEQ ID NO: 20 as recited in claims 21xxii and 22xxii.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 20 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim.
Claim 20 recites “an amino acid listed in Table 5”. Claims must be stand alone and must not refer to tables except for in exceptional circumstances (MPEP2173.05(s)). This is not an exceptional circumstance.
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.
Claims 17-36 are rejected under 35 U.S.C. 103 as being unpatentable over CHAN (Chan et al., Patent No: US 8,618,354 B2, Date of Patent: Dec. 31, 2013; included on IDS dated 08/27/2024) in view of SANEI (Sanei et al., 2011, PNAS, vol. 108(33), pp. E498-E505; included on IDS dated 08/27/2024).
Claim 17 recites “[a] plant having a biological activity of a haploid inducer and comprising a polynucleotide which comprises a nucleotide sequence encoding a centromeric histone H3 (CENH3) protein, wherein the nucleotide sequence comprises at least one introduced mutation that alters the amino acid sequence of the CENH3 protein in an α2-helix”.
CHAN teaches that CENH3 proteins are a well characterized class of proteins that are variants of H3 histone proteins and that are specialized proteins associated with the centromere. CENH3 proteins are characterized by a variable tail domain, which does not form a rigid secondary structure, and a conserved histone fold domain made up of three α-helical regions connected by loop sections (Chan, column 9, lines 8-14).
CHAN further teaches that CENH3 is a member of the kinetochore complex, the protein structure on chromosomes where spindle fibers attach during cell division. Accordingly, the present invention provides for plants, fungi, or animals (or cells thereof) that express a recombinant mutated kinetochore protein that disrupts the centromere, and/or plants, fungi, or animals (or cells thereof) in which at least one or both copies of an allele of the endogenous CENH3 gene has been knocked out, mutated to reduce or eliminate its function, or silenced (Chan, column 8, lines 36-60).
CHAN teaches a transgenic plant comprising a heterologous transgene expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a recombinantly altered CENH3 polypeptide (i.e., a nucleotide sequence encoding a centromeric histone H3 (CENH3) protein, wherein the nucleotide sequence comprises at least one introduced mutation that alters the amino acid sequence of the CENH3 protein), wherein in the event the recombinantly altered polypeptide is expressed in a first plant having a corresponding inactivated endogenous CENH3 gene and the first plant is crossed to a wildtype plant, at least 0.1% of resulting progeny are haploid (i.e., a plant having a biological activity of a haploid inducer) (Chan, column 1, lines 43-52).
CHAN does not explicitly teach wherein the nucleotide sequence comprises at least one introduced mutation that alters the amino acid sequence of the CENH3 protein in an a2-helix.
However, CHAN teaches that any number of mutations of CENH3 can be introduced into a CENH3 protein to generate a mutated CENH3 protein capable of generating haploid plants when expressed in a plant lacking, or having suppressed expression of, an endogenous CENH3 protein, and where the resulting transgenic plant is crossed to a plant expressing a wildtype CENH3 protein. Active mutated CENH3 proteins can be identified, for example, by random mutagenesis, by single or multiple amino acid targeted mutagenesis, by generation of complete or partial protein domain deletions, by fusion with heterologous amino acid sequences, or by combinations thereof (Chan, column 10, lines 35-46).
CHAN further teaches that the mutated CENH3 protein contains a CENH3 histone-fold domain identical to the CENH3 histone-fold domain of an endogenous CENH3 protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more amino acids (Chan, columns 10-11, lines 66-67 and 1-3).
CHAN teaches the CENH3 histone-fold domain with the α2-helix in the highlighted box (see Figure below) (Chan, Drawings, Figure 1).
[AltContent: rect]
PNG
media_image1.png
146
633
media_image1.png
Greyscale
CHAN teaches that centromere incompatibility was previously hypothesized to cause selective genome elimination in interspecies crosses, but it was not known how centromeres could be manipulated to achieve this. A practical basis was established for engineering genome elimination by altering CENH3, a protein essential for centromere function in all eukaryotes. The fact that haploids were produced with both GFP-tailswap and GFP-CENH3 transgenes suggests that multiple different alterations to the protein may induce genome elimination in other plants. A cenh3 mutation or a gene silencing method such as RNA interference could be used to reduce or eliminate endogenous CENH3 function in a novel species (Chan, column 26, lines 39-58).
CHAN teaches that genome elimination induced by changes in CENH3 probably occurs during the first few zygotic mitoses, when centromeres from the two parents are loaded with different populations of CENH3 proteins. Expression of both wild-type and mutant CENH3 genes in subsequent cell cycles should rapidly equalize the amount of the two proteins in individual centromeres. Subtle differences in centromere DNA binding, kinetochore assembly, or coupling to spindle microtubules may be sufficient to slow the segregation of chromosomes containing altered CENH3, resulting in genome elimination. Cell cycle checkpoints in plants must be relaxed enough to allow wild type and mutant chromosomes to segregate differentially, and presumably to permit cytokinesis without complete chromosome segregation (Chan, column 27, lines 11-29).
Furthermore, SANEI teaches that chromosome elimination of one parental genome after fertilization of the egg by the sperm of another species is a fairly common phenomenon and results in the formation of haploid embryos. It has been exploited for barley and other species (e.g., wheat, potato) to produce doubled haploids for breeding and mapping purposes. The advantage of doubled haploids for breeders is that homozygosity can be achieved in the first generation, whereas in breeding systems, such as pedigree or backcrossing, several selfed generations are needed to obtain high levels of homozygosity (Sanei, page E498, left column, second paragraph).
SANEI further teaches that to test whether parent-specific inactivation of centromeres is involved in the mitosis-dependent process of chromosome elimination in interspecific hybrids, the centromere-specific histone H3 variant (CENH3) in chromosomally unstable and stable Hordeum vulgare × H. bulbosum combinations was analyzed. CENH3 was selected because in mammals, Caenorhabditis elegans, and Drosophila melanogaster, its loss results in the failure of centromere formation and chromosome segregation. A region in CENH3 defined as the centromere targeting domain (CATD) is critical for centromeric localization of CENH3 in various species. The CATD is composed of the loop1 linker and α2-helix of CENH3, and its substitution enabled the incorporation of an H3 chimera into centromeres. This domain mediates molecular recognition events before and after nucleosome assembly and is important for binding of CENH3 to centromeric DNA, to CENH3-specific chaperones, and to CENH3-stabilizing factors (Sanei, page E498, left column, last paragraph).
SANEI also teaches the CENH3 histone-fold domain with the α2-helix (see Figure below) (Sanei, Supporting Information, Figure S3A).
PNG
media_image2.png
222
565
media_image2.png
Greyscale
SANEI teaches that although centromeric DNA sequences are extremely diverse, all eukaryotic centromeres contain CENH3. The chromosomal location of CENH3 is the assembly site for the kinetochore complex of active centromeres. Any error in histone gene transcription, translation, modification, or import could affect the ability to assemble intact CENH3 chromatin, which would result in the loss of CENH3 from centromeres and, hence, of centromere identity (Sanei, page E498, right column, first full paragraph).
EXAMINER’S NOTE: It is noted that it is the position of this Office that any mutation introduced in any CENH3 gene location that results in the failure of centromere formation and chromosome segregation is rendered obvious by the teachings of CHAN and SANEI.
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to introduce a mutation in the CENH3 gene to disrupt centromere formation and chromosome segregation as taught by CHAN and SANEI. One of ordinary skill in the art would have anticipated that targeting the CENH3 gene would disrupt centromere formation and chromosome segregation as taught by CHAN and SANEI. One would have been motivated to combine the teachings of CHAN and SANEI knowing that the disruption of centromere formation and chromosome segregation would lead to the generation of haploid plants as taught by CHAN and SANEI. Thus, one of ordinary skill in the art would have a high expectation of success by combining the teachings of CHAN and SANEI.
In regard to claim 18, CHAN teaches that a “mutated” sequence refers to a human-altered sequence. Examples of human-induced mutation include exposure of an organism to a high dose of chemical, radiological, or insertional mutagen for the purposes of selecting mutants, as well as recombinant alteration of a sequence. Examples of human-induced recombinant alterations can include, e.g., fusions, insertions, deletions, and/or changes to the sequence (i.e., wherein the at least one mutation is a point mutation, an insertion or deletion of at least one nucleotide, a mutation in a splicing site, a substitution of one or more amino acids, an insertion of one or more amino acids or a deletion of one or more amino acids) (Chan, column 4, lines 55-62).
In regard to claims 19, 20, and 33, CHAN teaches that the mutated CENH3 protein is identical to an endogenous CENH3 protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more amino acids. For example, in some embodiments, the endogenous wildtype protein from the plant is identical or substantially identical to SEQ ID NOs: 10 or 16 and the mutated CENH3 protein differs from the endogenous CENH3 protein by 1, 2, 3, 4, 5, 6, 7, 8, or more amino acids (i.e., wherein the at least one mutation alters the amino acid sequence of the CENH3 protein in the a2-helix corresponding to nucleotides from position 379 to position 465 set forth in SEQ ID NO: 10 of the CENH3 protein derived from Arabidopsis thaliana set forth in SEQ ID NO: 11 (instant claims 19 and 33); wherein the at least one mutation causes an amino acid substitution or deletion of an amino acid of SEQ ID NO: 20 (instant claim 20)) (Chan, column 10, lines 58-64).
CHAN teaches Arabidopsis thaliana histone H3-like centromere protein (CENH3) (SEQ ID NO: 10) which shares 100% sequence identity with instant sequence SEQ ID NO: 11, the amino acid sequence of the wildtype A. thaliana CENH3 (see alignment below).
CHAN SEQUENCE SEQ ID NO: 10 ALIGNED WITH INSTANT SEQUENCE SEQ ID NO: 11
Qy 1 MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPERAAGTGGRAASGGDSVKKTKPRHRW 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPERAAGTGGRAASGGDSVKKTKPRHRW 60
Qy 61 RPGTVALREIRKYQKSTEPLIPFAPFVRVVRELTNFVTNGKVERYTAEALLALQEAAEFH 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RPGTVALREIRKYQKSTEPLIPFAPFVRVVRELTNFVTNGKVERYTAEALLALQEAAEFH 120
Qy 121 LIELFEMANLCAIHAKRVTIMQKDIQLARRIGGRRWA 157
|||||||||||||||||||||||||||||||||||||
Db 121 LIELFEMANLCAIHAKRVTIMQKDIQLARRIGGRRWA 157
CHAN teaches Zea mays centromeric histone H3 protein (CENH3) (SEQ ID NO: 16) which shares 100% sequence identity with instant sequence SEQ ID NO: 20, the amino acid sequence of the wildtype Zea mays CENH3 (see alignment below).
CHAN SEQUENCE SEQ ID NO: 16 ALIGNED WITH INSTANT SEQUENCE SEQ ID NO: 20
Qy 1 MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPERAAGTGGRAASGGDSVKKTKPRHRW 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPERAAGTGGRAASGGDSVKKTKPRHRW 60
Qy 61 RPGTVALREIRKYQKSTEPLIPFAPFVRVVRELTNFVTNGKVERYTAEALLALQEAAEFH 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RPGTVALREIRKYQKSTEPLIPFAPFVRVVRELTNFVTNGKVERYTAEALLALQEAAEFH 120
Qy 121 LIELFEMANLCAIHAKRVTIMQKDIQLARRIGGRRWA 157
|||||||||||||||||||||||||||||||||||||
Db 121 LIELFEMANLCAIHAKRVTIMQKDIQLARRIGGRRWA 157
In regard to claims 23 and 24, CHAN teaches a transgenic plant comprising a heterologous transgene expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a recombinantly altered CENH3 polypeptide (i.e., wherein the polynucleotide comprising the at least one mutation is an endogenous gene or a transgene (instant claim 24)), wherein in the event the recombinantly altered polypeptide is expressed in a first plant having a corresponding inactivated endogenous CENH3 gene and the first plant is crossed to a wildtype plant, at least 0.1% of resulting progeny are haploid (i.e., wherein crossing between the plant and a wildtype plant or a plant expressing wildtype CENH3 protein yields at least 0.1% haploid progeny (instant claim 23)) (Chan, column 1, lines 43-52).
In regard to claims 25 and 26, CHAN teaches that the term “plant” includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same (i.e., wherein the part is a leaf, a stem, a root, an emerged radicle, a flower, a petal, a fruit, pollen, a pollen tube, an anther filament, an ovule, an embryo sac, an egg cell, an ovary, a zygote, an embryo, a hypocotyl section, an apical meristem, a vascular bundle, a pericycle, a seed, a cutting, a cell culture, or a tissue culture (instant claim 25); wherein the part is a shoot, a vegetative organ, a root, a flower, a floral organ, a seed, a fruit, an ovule, an embryo, a plant tissue or a cell (instant claim 26)) (Chan, column 5, lines 9-16).
In regard to claim 27, CHAN teaches crossing plants that lack an endogenous kinetochore complex protein and express an active mutated kinetochore complex protein (e.g., a tailswap or other mutated CENH3 or non-CENH3 kinetochore complex protein) either as a pollen or ovule parent to a plant that expresses an endogenous kinetochore complex protein (e.g., CENH3 protein) will result in at least some progeny (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%, 20% or more) that are haploid and comprise only chromosomes from the plant that expresses the kinetochore complex protein. Thus, the present invention allows for the generation of haploid plants having all of its chromosomes from a plant of interest by crossing the plant of interest with a plant transgenically expressing the mutated kinetochore complex protein and collecting the resulting haploid seed (i.e., a method of generating a haploid plant, comprising the steps of: a) crossing the plant according to claim 17 to a plant expressing wildtype CENH3 protein; and b) identifying the haploid progeny plant generated from the crossing step) (Chan, column 21, lines 9-23).
In regard to claims 28 and 29, CHAN teaches that once generated, haploid plants can be used for a variety of useful endeavors, including but not limited to the generation of doubled haploid plants (i.e., converting the haploid progeny plant into a double haploid plant (instant claim 28c)).
Somatic haploid cells, haploid embryos, haploid seeds, or haploid plants produced from haploid seeds can be treated with a chromosome doubling agent. Homozygous double haploid plants can be regenerated from haploid cells by contacting the haploid cells with chromosome doubling agents, such as colchicine, anti-microtubule herbicides, or nitrous oxide to create homozygous doubled haploid cells (i.e., wherein in step c) the haploid progeny plant is converted into a double haploid plant via colchicine treatment (instant claim 29)) (Chan, column 21, lines 44-58).
In regard to claim 30, CHAN teaches that the plant expressing an endogenous wildtype CENH3 protein can be crossed as either the male or female parent. One unique aspect of the present invention is that it allows for generation of a plant having only a male parent's nuclear chromosomes and a female parent's cytoplasm with associated mitochondria and plastids, when the tailswap (mutation) parent is the male parent (i.e., a method of facilitating a cytoplasm exchange, comprising the steps of: a) crossing the plant according to claim 17 as an ovule parent with a plant expressing wildtype CENH3 protein as a pollen parent; and b) obtaining a haploid progeny plant comprising the chromosomes of the pollen parent and the cytoplasm of the ovule parent) (Chan, column 21, lines 24-30).
In regard to claims 31 and 32, CHAN teaches that haploid Arabidopsis thaliana can be easily generated through seeds by manipulating a single centromere protein, the centromere-specific histone CENH3/CENP-A (i.e., a method of generating a plant according to claim 17; a) subjecting seeds of a plant to a sufficient amount of a mutagen, to obtain M1 plants). When cenh3 null mutants expressing altered CENH3 proteins are crossed to wild type, chromosomes from the mutant are eliminated, producing haploid progeny. Haploids are spontaneously converted into fertile diploids through meiotic non-reduction, allowing their genotype to be perpetuated (i.e., b) allowing sufficient production of fertile M2 plants). Maternal and paternal haploids can be generated through reciprocal crosses. As CENH3 is universal in eukaryotes, our method can be extended to produce haploids in any plant species (i.e., c) isolating genomic DNA of M2 plants, and d) selecting individuals possessing at least one mutation in a polynucleotide comprising a nucleotide sequence encoding a centromeric histone H3 (CENH3) protein, wherein the at least one mutation causes an alteration of the amino acid sequence of the CENH3 protein in the N-terminal domain of CENH3 (instant claim 31)). (Chan, columns 22 and 23, lines 60-67 and 1-6).
CHAN teaches that cenh3-1 was isolated by the TILLING procedure. The TILLING population was created by mutagenizing Arabidopsis thaliana with ethylmethane sulfonate (i.e., wherein the mutagen is ethylmethane sulfonate (instant claim 32)) (Chan, column 27, lines 57-61).
In regard to claim 34, CHAN teaches that mutations in selected portions of a kinetochore complex protein gene sequences are made in vitro and then introduced into the desired plant using standard techniques. Since the efficiency of homologous recombination is known to be dependent on the vectors used, use of dicistronic gene targeting vectors are conveniently used to increase the efficiency of selecting for altered CENH3 gene expression in transgenic plants. The mutated gene will interact with the target wild-type gene in such a way that homologous recombination and targeted replacement of the wild-type gene will occur in transgenic plant cells, resulting in suppression of kinetochore complex protein activity (i.e., a vector comprising the polynucleotide of claim 33) (Chan, column 18, lines 30-45).
In regard to claims 35 and 36, CHAN teaches that a chimera was created in which the A. thaliana CENH3 tail from CENH3 is replaced with the CENH3 tail domain from maize (Zea mays), thereby generating a fusion of the maize CENH3 tail and A. thaliana CENH3 histone-fold domain, and transformed the fusion into cenh3-1 heterozygotes. When cenh3-1 GFP-maizetailswap females were crossed to wild-type males, 2 haploids, 3 diploids and 5 aneuploids were found among a total of 10 F1 progeny (i.e., a plant cell or a host cell comprising a polynucleotide comprising a nucleotide sequence encoding at least the N-terminal domain of the amino acid sequence of CENH3, wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequence of the at least one segment as a transgene or the vector of claim 34 as a transgene) (Chan, columns 28-29, lines 63-67 and 1-8).
Summary
No claim is allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CHRISTINA MEADOWS whose telephone number is (703)756-1430. The examiner can normally be reached Monday - Friday 9:00 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Amjad Abraham can be reached at 571-270-7058. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/CHRISTINA L MEADOWS/Examiner, Art Unit 1663
CHRISTINA MEADOWS
Examiner
Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663
Prosecution Insights