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 .
Claim Status
Claims 1-6, 9-12, 14-18, 21-27 are pending.
Claims 14-18 are withdrawn from examination as being part of a non-elected invention.
Claims 1-6, 9-12 and 21-27 are being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims. However, the claim amendments by the Applicant by adding new issues necessitated new prior art references and new grounds of rejections, as discussed below.
Claim Rejections - 35 USC § 112(a)
Written Description
Claims 1-6, 9-12 and 21-27 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are drawn to a method or a plant cell which require a transcriptional repressor polypeptide comprising at least 90% or 95% amino acid identity to SEQ ID NO: 181.
The Applicant does not give any example of a transcription repressor protein (TRBIP1) comprising less than 100% sequence identity to instant SEQ ID NO: 181 while claiming the broad genus of proteins having at least 90% (for claims 1, 21, dependents) or 95% (for claims 2 and 22) sequence identity to SEQ ID NO: 181.
The specification indicates that Table 19 discloses several TRBIP1 proteins from different plant species comprising SEQ ID NOs: 182-190 (page 61, para 0166, Table 19). However, the Applicant does not describe if all these proteins have at least 90% or 95% sequence identity with SEQ ID NO: 181. A quick sequence alignment between SEQ ID NO: 181 and SEQ ID NO: 182; and between SEQ ID NO: 181 and SEQ ID NO: 184, for example, shows that there is only 39.9% and 36.8%, respectively, sequence identity (data not shown) between the two proteins. The Applicant asserts that the polypeptides mentioned in Table 19 (page 61) are TPBIP1 polypeptides. But the specification does not provide any data that correlates the structures of the sequences of SEQ ID NOs: 182-190 with TRBIP1 functional activity. A quick search for the gene names of SEQ ID NOs: 182-190 (e.g., Gene Name: XP_003517132.1 comprising SEQ ID NO: 182) shows that the proteins are uncharacterized or hypothetical (data not shown).
Mutating 10% or 5% of 333 amino acid long SEQ ID NO: 181 along the entire length of the protein would allow mutating up to 33 and 16 amino acids, respectively. The Applicant does not describe any structure function relationship that would enable any skilled artisan to mutate up to 33 or 16 amino acid residues in SEQ ID NO: 181 while retaining the function to repress transcription of target gene(s) and fulfilling the limitations of claim 1.
Current status of the art also does not provide any structure function relationship that would enable any skilled artisan to mutate up to 66 amino acid in SEQ ID NO: 181 while retaining the function to repress transcription of target gene(s). Moreover, it is known in the art that mutating even a single amino acid, which might be crucial for its stability and/or any specific function of the polypeptide including protein-protein interaction(s) and/or polynucleotide-protein interaction(s) can drastically change the transcription repression function of the polypeptide.
Considering the breadth of the claims, lack of representative species of the broad genus claimed, lack of structure function relationship of the broad genus claimed, and unpredictability of the art, the Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Scope of enablement
Claims 1-6, 9-12 and 21-27 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 a transcriptional repressor polypeptide having 100% sequence identity to SEQ ID NO: 181, does not reasonably provide enablement for a transcriptional repressor polypeptide having at least 90% or 95% sequence identity to SEQ ID NO: 181. 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.
The invention is related to reducing expression (repression) of a target nucleic acid in plants using various (recombinant) proteins (spec, page 1, para 0003).
The claims are drawn to a method or a plant cell which require a transcriptional repressor polypeptide comprising at least 90% or 95% amino acid identity to SEQ ID NO: 181.
The Applicant does not give any working example of a transcription repressor protein (TRBIP1) comprising less than 100% sequence identity to instant SEQ ID NO: 181.
Mutating up to 10% or 5% of 333 amino acid long SEQ ID NO: 181 along the entire length of the protein would allow mutating up to 33 or 16 amino acid residues. The Applicant does not provide any guidance for any skilled artisan to mutate up to 33 or 16 amino acid residues in SEQ ID NO: 181 so that the mutated protein retains the function of a transcriptional repressor.
The specification describes several proteins from different plant species comprising SEQ ID NOs: 182-190 (page 61, para 0166, Table 19). However, the Applicant does not describe if all these proteins have at least 90% or 95% sequence identity among themselves or with SEQ ID NO: 181. A quick sequence alignment between SEQ ID NO: 181 and SEQ ID NO: 182; and between SEQ ID NO: 181 and SEQ ID NO: 184 shows that there is only 39.9% and 36.8%, respectively, sequence identity (data not shown) between the two proteins. The Applicant asserts that the polypeptides mentioned in Table 19 are TPBIP1 polypeptides. But the specification does not provide any data that correlates the structures of the sequences of SEQ ID NOs: 182-190 with TRBIP1 functional activity. A quick search for some of the gene names of SEQ ID NOs: 182-190 (e.g., gene name: XP_003517132.1 comprising SEQ ID NO: 182) shows that the proteins are uncharacterized or hypothetical (data not shown).
Current status of the art does not provide any guidance for any skilled artisan to mutate up to 33 or 16 amino acid residues in SEQ ID NO: 181 while the mutated protein retains the function of a transcriptional repressor. It is known in the art that mutating even a single amino acid, crucial for its stability and/or any specific function of the polypeptide including protein-protein interaction(s) and/or polynucleotide-protein interaction(s), can drastically change the function(s) of the polypeptide.
Undue trial and error experimentation would be needed to make a transcription repressor protein comprising at least 90% or 95% sequence identity to SEQ ID NO: 181 and maintains the function of a transcriptional repressor.
Based on breadth of the claims, lack of any working example, lack of guidance in the instant description or in prior art, the specification at the time of the application filed would not have taught one skilled in the art how to make and use the full scope of the claimed invention without performing undue experiments.
Claim Rejections - 35 USC § 103
Claims 1-6 and 21-27 are rejected under 35 U.S.C. 103 as being unpatentable over Mayer et al. (Nature, Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana, 1999, 402(6763):769-77) in view of Wu et al. (Genome wide analysis of PHD finger family in Soybean (Glycine max), 2014, Advanced Materials Research, 864-867:2503-2508) and Hiratsu et al. (Identification of the minimal repression domain of SUPERMAN shows that the DLELRL hexapeptide is both necessary and sufficient for repression of transcription in Arabidopsis, 2004, Biochemical and Biophysical Research Communications, 321:172–178).
Claims 1-2 and 21-22 are drawn to a method of producing a plant with reduced expression of a target polynucleotide (gene) by using a heterologous targeting domain and a transcriptional repressor polypeptide, TRBPI1, comprising an amino acid sequence having at least 95% amino acid identity to SEQ ID NO: 181 and a heterologous targeting domain.
Mayer et al. describes a PHD finger polypeptide having at least 95% (100%) sequence identity to instant SEQ ID NO: 181, data not shown. The same polypeptide with a locus tag At4g355101-3 has been renamed a few times and ended up renamed as TRB INTERACTING PROTEIN1 (TRBIP1)2. PHD finger proteins are known to regulate/repress transcription via modification of chromatin structure, as discussed below.
However, Mayer et al. does not explicitly describe a heterologous targeting domain.
Wu et al. describes plant homeobox domain (PHD) proteins comprising a relatively small motif comprising approximate 60 amino acids that have been found in more than 400 eukaryotic proteins (page 1, para 2, line 1-2) including Arabidopsis (page 1, para 2, line 5-6). The PHD domain is highly conserved and found in an increasing number of proteins with roles in regulating/repressing transcription via modification of chromatin structure (page 1, para 2, line 2-4). Wu et al. also describes that the PHD finger protein family comprises one or more DNA binding domains (as recited in claims 3 and 23) including DTT domain (page 2, para 3, line 13), AT-Hook domain which preferentially binds to preference for A/T rich regions in the DNA (page 2, para 3, line 18-19), and/or C2HC zinc finger domain that binds to RNA or single stranded DNA (page 2, para 3, last 3 lines). Many of those PHD finger proteins repress several genes including KAP-1, Trithorax-like protein (Tcl), and Polycomb-like protein (Pcl) (page 1, para 2, line 4-6). PHD finger domain can bind two zinc atoms (page 2503, para 3, line 3) and is considered as zinc finger (as recited in claims 4 and 24) as well. Naturally, all PHD finger domain are obtained by simply collecting all of the canonically spaced zinc fingers ((page 2504, para 2, line 4-5).
Hiratsu et al. describes an Arabidopsis plant (as recited in claim 27) expressing a recombinant protein encoded by a recombinant nucleic acid (as recited in claim 26) comprising the Repressor Domain (RD) from SUPRD gene fused with the DNA-binding domain (i.e., “targeting domain”) of the GAL4 (GAL4DB) from yeast (page 173, right column, para 1, line 15-19). The fusion protein functions as a (transcriptional) repressor (abstract; page 176, right column, para 2, line 1) repressing the expression of a reporter gene (page 176, right column, para 2, line 14-16; Fig. 4B).
Before the effective filing date of the invention, it would have been obvious to one with ordinary skill in the art to make a recombinant polypeptide (as recited in claim 6) by fusing a transcriptional repressor protein like a PHD protein TRBIP1, as described by Mayer et al. and Wu et al., with a DNA binding targeting domain such as that of GAL4, as described by Hiratsu et al., with a realistic objective to repress expression of specific target gene(s) based on the specificity of the (DNA binding) targeting domain, as described by Hiratsu et al. and Wu et al. Using the DNA binding domain of the PHD finger protein described by Wu et al. would enable the artisan to repress KAP-1, Trithorax-like protein (Tcl), and Polycomb-like protein (Pcl), as taught by Wu et al. On the other hand, using GAL4 or other DNA binding domain would enable the artisan to repress other target gene(s), as described by Hiratsu et al.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to use TRBIP1(locus tag At4g35510) with the realistic objective to repress specific target gene(s) by fusing the PHD finger like protein TRBIP1 with a heterologous DNA binding targeting domain of Zinc finger as found in many PHD finger proteins. On the other hand, using GAL4 or other DNA binding domains would enable the artisan to repress other target gene(s).
Regarding claims 5 and 25; use of the “wherein” clause in the method claims 5 and 25 imply that reduction of target gene expression by at least 50% is the intended result of a process step positively recited. The intended result would have been the same or similar if an ordinarily skilled artisan would have followed the active steps based on the teachings of Mayer et al. and Wu et al., as described above. Nonetheless, the intended result of reduction of target gene expression by at least 50% is not needed to decide patentability of the claims.
The Applicant is remined that a “whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.’" Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003). See MPEP § 2111.04.
Claims 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over Mayer et al. in view of Wu et al. and Hiratsu et al. as applied to claims 1-6 and 21-27 above, and further in view of Jacobsen et al. (US20190203216A1, published on 7/4/2019) and To et al. (DNA Methylation within Transcribed Regions, 2015, Plant Physiology, 168:1219–1225).
Claim 9 depends from claim 1, and is drawn to a recombinant DNA methyltransferase polypeptide which is capable of being targeted to the target nucleic acid.
Mayer et al. in view of Wu et al. and Hiratsu et al. describes a method for producing a plant with reduced expression of a target nucleic acid by using a transcriptional repressor polypeptide TRBIP1 and heterologous targeting domain, whereby the recombinant polypeptide is expressed and targeted to the target nucleic acid, thereby reducing expression of the target nucleic acid to produce the plant with reduced expression of the target nucleic acid, as discussed above.
However, Mayer et al. in view of Wu et al. and Hiratsu et al. does not describe any DNA methyltransferase polypeptide.
Jacobsen et al. describes a method for reducing expression (repressing) of a target nucleic acid in a plant by expressing a recombinant polypeptide comprising a DNA-binding domain capable of binding a target nucleic acid and a second amino acid sequence comprising a DNA methyltransferase polypeptide, and growing the plant under conditions whereby the recombinant polypeptide encoded by the recombinant nucleic acid is expressed and binds to the target nucleic acid, thereby reducing expression of the target nucleic acid (claim 1). Methylation of DNA and of specific proteins including histone H3 by DNA methyltransferase are important for formation of heterochromatin and transcriptional gene silencing (page 1, para 0004, line 10-13). Jacobsen et al. also describes a transgenic plant (page 62, para 0575, line 1-8) expressing a recombinant a DNA methyltransferase MQ1 protein produced using a plant codon-optimized cDNA sequence of the Methyltransferase gene from Spiroplasma sp. strain MQ1 (M.Sss1), as recited in claim 10. The MQ1 methyltransferase described by Jacobsen et al. comprises at least 80% (99.7%) sequence identity to instant SEQ ID NO: 212 (as recited in claim 11).
Before the effective filing date of the invention, it would have been obvious to one with ordinary skill in the art to modify Mayer et al. in view of Wu et al. and Hiratsu et al. by fusing the TRBIP1 protein with a MQ1 methyltransferase (M.Sss1) polypeptide, as described by Jacobsen et al. with a realistic objective to enhance efficiency of repression or silence the expression of specific target gene(s), as described by Jacobsen et al. The TRBIP1 protein would provide the DNA binding function in specific target gene(s). It is known in the art that high levels of DNA methylation are found only in silent genes and transposable elements (TEs) in plants (To et al.; page 1219, left column, line 5-7). In contrast, actively transcribed genes in plants do not have any or much methylation especially around its promoter region (To et al.; page 1219, left column, line 7-9). Binding of methyltransferases on to the promoter region(s) of the target gene(s) via TRBIP1 and resulting methylation would enhance the efficiency of repression which even would render the gene transcriptionally inactive.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to fuse a MQ1 polypeptide with the TRBIP1with a realistic objective to increase the efficiency to repress and/or silence expression of specific target gene(s).
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Mayer et al. in view of Wu et al. and Hiratsu et al. as applied to claims 1-6 and 21-27 above, and further in view of Afzal et al. (Seed Production Technologies of Some Major Field Crops, Agronomic Crops. Ed. by Mirza Hasanuzzaman. Singapore: Springer, 2019. 655–678).
Claims 12 depends from claim 1 and is drawn to crossing the plant with reduced expression of the target nucleic acid to a second plant to produce one or more F1 plants.
Mayer et al. in view of Wu et al. and Hiratsu et al. describes a method for producing a plant with reduced expression of a target nucleic acid by using a transcriptional repressor polypeptide TRBIP1 and growing the plant under (abiotic stress) conditions whereby the recombinant polypeptide is expressed and targeted to the target nucleic acid, thereby reducing expression of the target nucleic acid to produce the plant with reduced expression of the target nucleic acid, as discussed above.
However, Mayer et al. in view of Wu et al. and Hiratsu et al. does not describe crossing the plant with reduced expression of the target nucleic acid to a second plant to produce one or more F1 plants.
Afzal et al. describes introgressing different traits in a single variety/cultivar (transgressive population) by crossing a parental line with a second line (page 658, para 2, line 9-11). It is a standard practice in the art to cross a transgenic line with the desired trait with an established elite line (which reads on to “second plant”) producing one or more F1 plants. Establishing F1 population by crossing two different (inbred) lines to exploit heterosis is also a standard practice in the art to develop hybrid seeds (Afzal et al., page 676, para 2, line 2-4). Afzal et al. describes that selecting segregants in F2 to F5 generations would yield transgressive population which would be better than both the parents (page 658, para 2, line 10-11).
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to modify Mayer et al. in view of Wu et al. and Hiratsu et al. by crossing the transgenic plants with capability to repress specific target gene(s) under specific (cold) stress condition with a second plant to produce one or more F1 plants, as described by Afzal et al.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to cross the transgenic plants with capability to repress specific target gene(s) under specific (cold) stress condition with a second elite plant to produce one or more F1 plants with a realistic objective to introgress valuable traits belonging to the second parental line to obtain a transgressive population which would be better than both the parents.
Response to Applicant’s argument
In regard to claim rejections under 35 USC 112(a) (Written Description): The Applicant argues, “application as filed describes TRBIP1 polypeptides and provides exemplary homologs (with sequence information) from other species (See e.g. paragraphs [0163] - [0167] and Table 19 of the specification as filed). The specification as filed also characterizes TRBIP1 polypeptides as interacting with TRB proteins, and that TRBIP1 proteins are annotated as PHD finger-like proteins (See e.g. paragraphs [0163] - [0164]” (response, page 7, para 2, line 4-8). Applicant’s arguments have been fully considered but are not found persuasive. The specification describes several proteins from different plant species comprising SEQ ID NOs: 182-190 (page 61, para 0166, Table 19). The Applicant asserts that the polypeptides mentioned in Table 19 (page 61) are TPBIP1 polypeptides. But the specification does not provide any data that correlates the structures of the sequences of SEQ ID NOs: 182-190 with TRBIP1 functional activity. A quick search for some of the gene names of SEQ ID NOs: 182-190 (e.g., Gene Name: XP_003517132.1 comprising SEQ ID NO: 182) shows that the proteins are uncharacterized or hypothetical (data not shown). Moreover, the Applicant does not describe if all these proteins have at least 90% or 95% sequence identity among themselves or with SEQ ID NO: 181. A quick sequence alignment between SEQ ID NO: 181 and SEQ ID NO: 182; and between SEQ ID NO: 181 and SEQ ID NO: 184 shows that there is only 39.9% and 36.8%, respectively, sequence identity (data not shown) between the two proteins.
The Applicant describes TRBIP1 proteins as proteins that interact with TRB proteins and cite the example is only from the Arabidopsis protein (Spec, para 0164), i.e., SEQ ID NO: 181. The instant specification itself asserts that “endogenous function of TRBIP1 proteins have not been elucidated” (page 60, para 0164, last 2 lines). It is known in the art that TRB family of proteins interact with several protein complexes and each complex comprise many proteins. Most of the TRB interacting proteins are not TRBIP1 but telomerase, Polycomb repressive complex 2 (PRC2), E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs), EMF2, and VRN2 (Kusová et al., Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions, 2023, Plant Molecular Biology, 112:61–83; abstract).
Conclusion
All claims are rejected.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 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, Bratislav Stankovic can be reached at (571) 270-0305. 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.
Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 PHD finger-like protein (Arabidopsis thaliana), GenBank Accession No. AEE86524.1
2 Wang et al. ( Histone H3 lysine 4 methylation recruits DNA demethylases to enforce gene expression in
Arabidopsis, 2025, Nature Plants, 11:206–217; page 213, right column, last para, line 4-5).