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
1. Claims 1-4, 32-33, and 36-37 are pending and under examination to the extent of the elected species of DCL2, DCL4, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:1, and SEQ ID NO:3.
Claims 17-22, 24-26, and 31 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on May 09, 2025.
Claims 5-16, 23, 27-30, and 34-35 are cancelled.
Information Disclosure Statement
2. The information disclosure statement (IDS) submitted on October 16, 2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner. A signed copy is attached.
Response to Arguments – Objections to the Specification
3. Applicant’s arguments and amendments filed November 14, 2025 have been carefully considered but are not persuasive and do not overcome all of the objections of record.
Regarding Applicant’s failure to italicize the gene names of DCL2, DCL4, RDR1, RDR2, and RDR6 and the species names of Arabidopsis and Aloe vera in the specification filed on April 04, 2023, it appears Applicant has attempted to amend said gene and species names to be italicized in the substitute specification filed on November 14, 2025. However, said amendments have not been entered properly. 37 C.F.R. 1.121(b)(ii) dictates that the text of any added subject matter must be shown by underlining the added text; the text of any deleted matter must be shown by strike-through except that double brackets placed before and after the deleted characters may be used to show deletion of five or fewer consecutive characters; and the text of any deleted subject matter must be shown by being placed within double brackets if strike-through cannot be easily perceived. Applicant did not demonstrate the deletion of the non-italicized names by either strike-through or double-bracketing, nor did Applicant demonstrate the addition of subject matter via underlining the added subject matter. Accordingly, these amendments are improper and are not entered; therefore, these objections are maintained.
Objections to the Specification
4. The specification of the disclosure remains objected to because of the following reasons:
On p. 03, ln. 11, “DCL2, DCL4, RDR1, RDR2, and RDR6” should be italicized because it is standard in scientific notation to italicize the names of genes. All subsequent recitations of the genes “DCL2", “DCL4”, “RDR1”, “RDR2”, and “RDR6” should likewise be italicized.
On p. 06, ln. 14, “Arabidopsis”, and “Aloe Vera” should be italicized because it is standard in scientific notation to italicize formal genus and species names. See also: p. 13, ln. 34 and p. 14, lns. 1-30. Furthermore, the “vera” should be lower-cased.
Appropriate correction is required.
Response to Arguments - Claim Objections
5. Applicant’s arguments and amendments filed November 14, 2025 have been carefully considered but are not persuasive and do not overcome all of the objections of record.
Regarding Applicant’s failure to italicize the gene names of DCL2, DCL4, RDR1, RDR2, and RDR6 in the claims filed on April 04, 2023, it appears Applicant has attempted to amend said gene and species names to be italicized in the amended claims filed on November 14, 2025. However, said amendments have not been entered properly. 37 C.F.R. 1.121(b)(ii) dictates that the text of any added subject matter must be shown by underlining the added text; the text of any deleted matter must be shown by strike-through except that double brackets placed before and after the deleted characters may be used to show deletion of five or fewer consecutive characters; and the text of any deleted subject matter must be shown by being placed within double brackets if strike-through cannot be easily perceived. Applicant did not demonstrate the deletion of the non-italicized names by either strike-through or double-bracketing, nor did Applicant demonstrate the addition of subject matter via underlining the added subject matter. Accordingly, this amendment is improper and is not entered; therefore, this objection is maintained.
Furthermore, Applicant’s amendments filed November 14, 2025 have necessitated new grounds of objections to the claims.
Claims 34-35 are cancelled; therefore, any objections to these claims have been rendered moot.
Claim Objections
6. Claims 1-4, 32-33, and 36-37 are objected to because of the following informalities:
In claim 1 and all subsequent recitations regarding a gene name, “DCL2”, “DCL4”, “RDR1”, “RDR2”, and “RDR6” should be italicized because it is standard in scientific notation to italicize the names of genes. See: claims 2-4 and 33.
Claim 1, ln. 3 contains a grammatical error; it is recommended Applicant insert the article “the” before “DCL2”.
Dependent claims are included.
Appropriate correction is required.
Response to Arguments - Claim Rejections - 35 USC § 112(b)
7. Applicant’s arguments and amendments filed November 14, 2025 have overcome the rejections of record. However, said amendments have necessitated new grounds of rejections.
Response to Arguments - Claim Rejections - 35 USC § 103
8. Applicant’s arguments and amendments filed November 14, 2025 have been carefully considered but are not persuasive and do not overcome all of the rejections of record.
In traversing the rejection of claims 1-4 and 34-37 under 35 U.S.C. 103 as being unpatentable over Matsuo et al. (Journal of Bioscience and Bioengineering. 2017: 124(2):215-220 (previously cited)) in view of Häkkinen et al. (Frontiers in Plant Science. 2018; 9:45 (previously cited)), Applicant argues that Matsuo is limited to RNA silencing and does not disclose or suggest tobacco cells in suspension nor the simultaneous knockout of all alleles of DCL2 and DCL4 and expresses an alleged preference for an alternative strategy of transcriptional repression instead of gene disruption (Applicant’s remarks dated 11/14/2025, pp. 09-10). Applicant additionally argues that Häkkinen also does not disclose or suggest knockout of DCL2 and DLC4 genes in isolated tobacco suspension cells (Applicant’s remarks dated 11/14/2025, p. 10).
Regarding Applicant’s assertion that Matsuo is limited to RNA silencing and does not disclose or suggest the simultaneous knockout of all alleles of DCL2 and DCL4, the Office respectfully disagrees. First, Matsuo provides motivation to disrupt the expression of both DCL2 and DCL4 in teaching that simultaneously reducing the expression of both genes results in greater recombinant protein expression than reducing the expression of either DCL2 or DCL4 alone (p. 217, Transient expression of human aFGF in NbDCL2- and NbDCL4-repressed plants”). Second, the RNAi-based gene silencing method disclosed by Matsuo is prima facie equivalent to the instant DCL2 and DCL4 loss-of-function mutations because the prior art element disclosed by Matsuo performs the same function (i.e., reduction of DCL2 and DCL4-mediated DICER activity) in substantially the same way (i.e., inhibition of DCL2 and DCL4 gene expression) and produces substantially the same results (i.e., increased expression of heterologous/recombinant proteins; see: MPEP 2183). Matsuo efficiently reduces expression of DCL2 and DCL4 to 20% and 25% of the wildtype expression levels, respectively (Matsuo; p. 217, “Production of DCL-repressed N. benthamiana plants”; Figure 2), which results in up to a 400% increase in the expression of recombinant proteins in comparison to wildtype plants (p. 217, Transient expression of human aFGF in NbDCL2- and NbDCL4-repressed plants”). Matsuo’s RNAi-based repression of DCL2 and DCL4 expression yields the same result as Applicant’s recited method (i.e., repression of DCL2/DCL4-based gene silencing). Various methods of gene silencing are well-known in the art, the selection of any particular method (e.g., RNAi or gene editing) is a routine and trivial design choice parameter well within the means of one of ordinary skill in the art, and Applicant has made no assertion and demonstrated no evidence of surprising and/or unexpected results that would distinguish the results or effects of the claimed method from those established in the prior art (see MPEP 716.02 and MPEP 2144.04). Matsuo even directly teaches RNA silencing as a form of gene knockout in contrast to Applicant’s assertion that does not suggest the simultaneous knockout of DCL2 and DCL4 (p. 216, left column, first full paragraphs, lns. 1-4). Thus, the RNAi-based gene silencing disclosed by Matsuo is prima facie equivalent to the instant loss-of-function mutations. Therefore, one of ordinary skill in the art would be motivated by the teachings of Matsuo to reduce the expression of DCL2 and DCL4 in tobacco cells, would have a reasonable expectation of success in reducing said expression via either RNAi-based gene silencing or targeted gene editing, and would recognize that the selection of either technique was simply matter of preference among alternatives (as suggested by Applicant on p. 10 of the remarks dated 11/14/2025).
In response to applicant's piecemeal analysis of the references, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The combined teachings, suggestions, and motivations of the cited references must be considered in their totality. Though Matsuo may be silent to tobacco suspension cells, Häkkinen teaches said cells and provides motivation for one of ordinary skill in the art to use tobacco suspension cells for recombinant protein expression (Häkkinen; Abstract; p. 01, first paragraph; p. 07, fourth full paragraph). Similarly, Matsuo teaches plants and tobacco cells as an attractive platform for the production of recombinant proteins and that inhibition of endogenous gene silencing mechanisms (e.g., DCL2 and DCL4) would increase protein expression in plant-based expression systems which would increase the utility of said systems (Abstract; p. 215, first paragraph; p. 216, first full paragraph). Furthermore, the generation and use of tobacco suspension cell cultures for recombinant protein expression is routine in the art. Thus, the combination of Matsuo and Häkkinen teaches and provides motivation for one of ordinary skill in the art to inhibit expression of DCL2 and DCL4 in tobacco suspension cells to improve the utility of a suspension cell culture for recombinant protein expression.
For the reasons discussed above, the rejection of claims 1-4 and 34-37 under 35 U.S.C. 103 as being unpatentable over Matsuo et al. (Journal of Bioscience and Bioengineering. 2017: 124(2):215-220) in view of Häkkinen et al. (Frontiers in Plant Science. 2018; 9:45 (previously cited)) is maintained.
Applicant’s amendments to claim 1 have successfully overcome the rejection of claims 1-4 and 32-34 under 35 U.S.C. 103 as being unpatentable over Moissiard et al. (RNA. 2007; 13:1268-1278 (previously cited)) in view of Rivero et al. (Methods in Molecular Biology. 2014; 1062:3-25 (previously cited)). However, said amendments have necessitated new grounds for rejection under 35 U.S.C. 103.
Claims 34-35 are cancelled; therefore, any rejections to these claims are rendered moot.
Claim Rejections - 35 USC § 103
9. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
10. Claims 1-4 and 36-37 are rejected under 35 U.S.C. 103 as being unpatentable over Matsuo et al. (Journal of Bioscience and Bioengineering. 2017: 124(2):215-220 (previously cited))) in view of Häkkinen et al. (Frontiers in Plant Science. 2018; 9:45 (previously cited)).
Regarding claim 1, Matsuo teaches an isolated Nicotiana benthamiana tobacco plant cell comprising reduced expression of all alleles of DCL2 and DCL4 in said plant cell (Abstract; p. 217, “Production of DCL-repressed N. benthamiana plants”), that plants provide advantages for the production of recombinant proteins due to increased safety and lower costs (Abstract; p. 215, first paragraph), and that inhibition of endogenous RNA silencing mechanisms improves recombinant protein expression in plants (Abstract; p. 216, first full paragraph).
Matsuo does not teach a plant cell in suspension and is silent to loss-of-function mutations in DCL2 and DCL4.
However, Häkkinen teaches wild-type and transgenic N. tabacum BY-2 suspension cell cultures (Abstract; p. 02 “Cell Lines”) and that BY-2 suspension cell cultures are an attractive platform for recombinant protein production (Abstract; p. 01, first paragraph; p. 07, fourth full paragraph).
The combination of Matsuo and Häkkinen teaches an isolated N. tabacum BY-2 tobacco plant cell in suspension comprising reduced expression of all alleles of DCL2 and DCL4 in said plant cell.
The level of ordinary skill in the plant biotechnology art is high as evidenced by both Matsuo and Häkkinen. It would have been prima facie obvious for one of ordinary skill in the art to introduce the RNAi constructs taught by Matsuo into the suspension cell culture taught by Häkkinen, because: 1) growing cells in suspension culture is routine in the art; 2) Matsuo teaches that inhibition of DICER-based RNA silencing improves the expression of recombinant proteins in tobacco cells; 3) Häkkinen teaches tobacco BY-2 cell line suspension cultures as an attractive platform for protein expression; 4) and both Matsuo and Häkkinen teach tobacco plants as an attractive system for producing recombinant proteins. Thus, one of ordinary skill in the art would be motivated by the teachings of Matsuo to reduce the expression of DCL2 and DCL4 in the tobacco BY-2 cell line suspension cultures taught by Häkkinen to produce a suspension cell culture capable of producing increased amounts of recombinant proteins at a lower cost with increased safety. Additionally, various methods of gene silencing are well-known in the art and the selection of any particular method is a routine and trivial design choice parameter well within the means of one of ordinary skill in the art. The RNAi-based repression of DCL2 and DCL4 expression taught by Matsuo yields the same result as Applicant’s recited method (i.e., repression of DCL2/DCL4-based gene silencing) and Applicant has made no assertion and demonstrated no evidence of surprising and/or unexpected results that would distinguish the results, effects, or function of the claimed invention from those established in the prior art (see MPEP 716.02 and MPEP 2144.04). Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results.
Regarding claims 2-4, Matsuo is silent to the region of the DCL2 and DCL4 alleles containing the loss of function mutations. However, as stated above, the generation of loss-of-function mutations (and other means of reducing gene expression) is routine in the art. The location of a mutation within a gene is a design choice parameter well within the means of one of ordinary skill in the art. The specific location of the mutation causing reduced gene activity does not yield any surprising or unexpected effect on gene activity regardless of the location of the mutation absent evidence or assertion of some surprising or unexpected result. Therefore, the combined teachings of Matsuo and Häkkinen satisfy these claim limitations. See MPEP 716.02 and MPEP 2144.04.
Regarding claim 36, the combined teachings of Matsuo and Häkkinen are as discussed above. Matsuo does not teach N. tabacum. However, Häkkinen teaches N. tabacum BY-2 suspension cell cultures (Abstract; p. 02 “Cell Lines”).
Regarding claim 37, the combined teachings of Matsuo and Häkkinen are as discussed above. Matsuo does not teach a tobacco BY-2 cell line. However, Häkkinen teaches N. tabacum BY-2 suspension cell cultures (Abstract; p. 02 “Cell Lines”).
Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results.
11. Claims 1-4, 32-33, and 36-37 are rejected under 35 U.S.C. 103 as being unpatentable over Matsuo et al. (Journal of Bioscience and Bioengineering. 2017: 124(2):215-220 (previously cited))) in view of Moissiard et al. (RNA. 2007; 13:1268-1278 (previously cited)), and further in view of Häkkinen et al. (Frontiers in Plant Science. 2018; 9:45 (previously cited).
Regarding claim 1, Matsuo teaches an isolated N. benthamiana tobacco plant cell comprising reduced expression of DCL2 and DCL4 in said plant cell (Abstract; p. 217, “Production of DCL-repressed N. benthamiana plants”), that plants provide advantages for the production of recombinant proteins due to increased safety and lower costs (Abstract; p. 215, first paragraph), and that inhibition of endogenous RNA silencing mechanisms improves recombinant protein expression in plants (Abstract; p. 216, first full paragraph).
Matsuo does not teach a plant cell in suspension or loss-of-function mutations in DCL2 or DCL4.
However, Moissiard teaches an isolated Arabidopsis plant cell comprising loss of function mutations in all alleles of DCL2 and DCL4 in said plant cell (p. 1273, Figure 4; p. 1274, left column, first full paragraph; p. 1276, left column, “Plant material”) and that inhibition of RNA silencing genes promotes expression of foreign and transgenes (Abstract; p. 1269, right column, first and second paragraph; p. 1270, Fig. 1).
Additionally, Häkkinen teaches wild-type and transgenic N. tabacum BY-2 suspension cell cultures (Abstract; p. 02 “Cell Lines”) and that BY-2 suspension cell cultures are an attractive platform for recombinant protein production (Abstract; p. 01, first paragraph; p. 07, fourth full paragraph).
The combination of Matsuo, Moissiard, and Häkkinen teaches an isolated tobacco plant cell in suspension comprising loss of function mutations in all alleles of the DCL2 and DCL4 genes in said tobacco plant cell.
The level of ordinary skill in the plant biotechnology art is high as evidenced by Matsuo, Moissiard, and Häkkinen. It would have been prima facie obvious for one of ordinary skill in the art to modify the tobacco suspension cells taught by Häkkinen to comprise the reduced expression and/or loss-of-function mutations of DCL2 and DCL4 as taught by Matsuo and Moissiard. One would have been motivated to do so because both Matsuo and Moissiard teach that reducing expression of DCL2 and DCL4 promotes the expression of recombinant proteins. One of ordinary skill would have been motivated to reduce the expression of DCL2 and DCL4 in suspension cells because: 1) increasing the production of transgenes and the proteins encoded therein would yield major improvements to the use of plant cell cultures for the heterologous production of pharmaceuticals; 2) the use of plant cell suspension cultures for protein production is routine in the art as demonstrated by Häkkinen; 3) both Matsuo and Moissiard teach that inhibition of DCL2 and DCL4 increases expression of transgenes; and 4) Matsuo’s and Moissiard’s similar teachings regarding the effects of silencing DCL2 and DCL4 in tobacco and Arabidopsis, respectively, suggests that reducing the expression of these genes would yield the same effect in any plant. Therefore, one of ordinary skill in the art would have been motivated to engineer a suspension cell culture consisting of plant cells comprising mutations that abolish the expression of DCL2 and DCL4 in a plant cell suspension culture.
Though Matsuo and Moissiard achieved similar results using distinct but functionally equivalent methods, one of ordinary skill in the art would be capable of modifying the expression of tobacco DLC2 and DCL4 by either the method of Matsuo or Moissiard because the development and use of RNAi constructs and the introduction of loss-of-function mutations are routine and well-known in the art. Various methods of gene silencing are well-known in the art, the selection of any particular method (e.g., RNAi or gene editing) is a routine and trivial design choice parameter well within the means of one of ordinary skill in the art, and despite their differing methods of reducing gene expression in different plant systems, both Matsuo and Moissiard reported similar results (i.e., reduced expression of DCL2 and DCL4 and increased expression of transgenes). Furthermore, Applicant has made no assertion and demonstrated no evidence of surprising and/or unexpected results that would distinguish the results or effects of the claimed method from those established in the prior art (see MPEP 716.02 and MPEP 2144.04). Thus, the RNAi-based gene silencing disclosed by Matsuo is prima facie equivalent to the instant loss-of-function mutations (see MPEP 2183). Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results.
Regarding claims 2-4, the teachings of Matsuo, Moissiard, and Häkkinen are as discussed above. Matsuo is silent to the region of the DCL2 and DCL4 alleles containing the loss of function mutations. However, as stated above, the generation of loss-of-function mutations (and other means of reducing gene expression) is routine in the art. The location of a mutation within a gene is a design choice parameter well within the means of one of ordinary skill in the art. The specific location of the mutation causing reduced gene activity does not yield any surprising or unexpected effect on gene activity regardless of the location of the mutation absent evidence or assertion of some surprising or unexpected result. Absent evidence or assertion of some surprising or unexpected result, loss-of-function mutations simply cause reduced protein activity regardless of the location of the mutation. Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results. See MPEP 716.02 and MPEP 2144.04.
Regarding claim 32, the teachings of Matsuo, Moissiard, and Häkkinen are as discussed above. Matsuo does not teach loss-of-function mutations and is silent to the method by which the expression of DCL2 and DCL4 are determined. However, as stated above, Moissiard teaches plant cells wherein said loss-of-function mutations abolish expression of said at least two genes. Additionally, the method by which the expression of a gene is assessed does not affect the expression of said gene and would not modify the gene activity of the cell. The means by which the expression of a gene is measured is simply a design parameter well within the means of one of ordinary skill in the art. Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results.
Regarding claim 33, the teachings of Matsuo, Moissiard, and Häkkinen are as discussed above. Matsuo is silent to siRNAs. However, Moissiard teaches plant cells wherein said loss-of-function mutations abolish expression and/or activity of said DCL2 and DCL4, as determined by no expression of transgene-specific 21-nt and 22-nt siRNAs in said plant cell following expression of said transgene in said plant cell (p. 1273, Fig. 4; p. 1273, left column, first full paragraph – p. 1274, left column, first full paragraph).
Regarding claims 36-37, the teachings of Matsuo, Moissiard, and Häkkinen are as discussed above. Matsuo does not teach a N. tabacum (claim 36) or BY-2 line cells (claim 37) and is silent to loss of function mutations in DCL2 and DCL4. However, Häkkinen teaches N. tabacum BY-2 line cells (Abstract; p. 02 “Cell Lines”).
Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results.
Conclusion
12. No claim is 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.
Examiner’s Contact Information
13. Any inquiry concerning this communication or earlier communications from the examiner should be directed to DEQUANTARIUS JAVON SPEED whose telephone number is (703)756-4779. The examiner can normally be reached M-F; 9AM-5PM ET.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Amjad Abraham can be reached on (571)-270-7058. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/DEQUANTARIUS JAVON SPEED/Junior Examiner, Art Unit 1663 /Amjad Abraham/SPE, Art Unit 1663