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 .
This is a Final Office Action in response to amendment filed on 7/20/2026.
Claim Status
Claims 1-7, 9-12, and 14-19 are pending. Claims 1, 4, and 10 are currently amended. Claims 8 and 13 are currently canceled. Claims 1-7, 9-12, and 14-19 are examined on the merits.
Claim Objections
Claims 1, 4 and 10 are objected to as not being properly marked to show the amendments in accordance with 37 CRR 1.121(c ).
Response to Amendment
The objection of Claim 10 is withdrawn in view of amendment to the claims.
The rejection of Claims 1-19 rejection under 35 U.S.C. 112(b) is withdrawn in view of amendment to the claims.
The rejection of claims 8 and 13 rejected under 35 U.S.C. §103 is withdrawn in view of the cancellation of those claims. The rejection of claims 1-7, 9-12, and 14-19 rejected under 35 U.S.C. §103 is modified in view of the amendments to claims 1, 4, and 10.
Applicant amended claim 1 by deleting the alternative limitation that the coding sequence may be “expressed at a low level”, such that claim 1 now requires that the coding sequence is “not expressed” before activation. This amendment narrows the scope of the claimed 1 by requiring the absence of expression before activation. Because this amended limitation was not addressed in the prior rejection and requires a different obviousness analysis, the rejection of claim 1 under 35 U.S.C. §103 is made as a new ground of rejection. The rejection of claims 2-7, 9-12, and 14-19, which depend directly or indirectly from claim 1, is likewise made as a new ground of rejection under 35 U.S.C. §103.
Claim Rejections - 35 USC § 112
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 15 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.
Claim 15 depends from claim 1 and recites that “the coding sequence is expressed at a higher level that is 1% - 100% higher than the initial low-level expression of the coding sequence”. However, amended claim 1 requires that, prior to activation by the site-specific promoter activation complex, “the coding sequence is not expressed”.
It is therefore unclear what constitutes the “initial low-level expression” recited in claim 15 because claim 1, form which claim 15 depends, does not establish any initial low-level expression. Rather, claim 1 expressly requires an absence of expression. The claim consequently provides no clear baseline from which the recited 1%-100% increase is to be calculated.
Moreover, it is unclear whether claim 15 is intended to modify the “not expressed” limitation of claim 1 to permit some initial expression, or whether the “initial low-level expression” refers to a different expression state not otherwise identified in the claim. Accordingly, the scope of claim 15 is unclear.
Claim Rejections - 35 USC § 112
Claim 15 rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 15 depends from claim 1, which requires that the coding sequence is “not expressed” before activation by the site-specific promoter activation complex. Claim 15, however, recites that the coding sequence has an “initial low-level expression” and that the subsequently expressed level is 1%-100% higher than that initial low-level expression.
The recitation of an initial low-level expression is inconsistent with, rather than further limitation of, the requirement in claim 1 that then adds a further limitation thereto. Instead, claim 15 appears to replace or modify the “not expressed” limitation of claim 1 with a different requirement permitting low-level expression.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-7, 14, and 18-19 are rejected under 35 U.S.C. §103 as being unpatentable over Hou (Zhenglin Hou, et. al., US20200190494A1, Application: 2019-12-13, Publication: 2020-06-18) in view of Kloti (Andreas S. Kloti, US6331663B1, Application: 1999-12-13, Publication: 2001-12-18).
Claim 1 recites a method of editing a genome of a target cell to comprise a donor polynucleotide, the method comprising: (a) Inserting a donor polynucleotide within a genomic target site, wherein the donor polynucleotide comprises a gene expression cassette comprising a non-functional promoter operably linked to a coding sequence such that the coding sequence is not expressed; (b) Binding a site specific-promoter activation complex to a genomic region upstream of the inserted donor polynucleotide, wherein the site specific-promoter activation complex functions to drive the non-functional promoter to express a protein from the coding sequence; (c) Expressing the coding sequence at a higher level; (d) Selecting for the target cell that expresses the coding sequence; and (e) Obtaining the target cell that expresses the coding sequence.
As an initial matter, the term “non-functional promoter” is interpreted under the broadest reasonable interpretation in view of the specification. The instant specification states that the “non-functional promoter” may comprise a minimal promoter (paragraph 0193). Accordingly, the claimed “non-functional promoter” is reasonably interpreted as encompassing a promoter that has no substantial independent expression activity, in the absence of upstream activating elements, but capable of being activated when such elements are provide.
Hou teaches insertion of a donor polynucleotide within a genomic target site in the genome of a cell using a Cas system (paragraph 0593, 0693), corresponding to step (a). Hou teaches that the donor polynucleotide may comprise a coding sequence including a selectable marker or agronomic trait sequence (paragraph 0502, 0607). Hou further teaches obtaining cells or organisms containing the inserted donor sequence (paragraph 0593), corresponding to steps (d) and (e). Hou additionally teaches Cas fusion proteins having transcription activation domains such as VP16 or VP64, and Cas complexes that bind DNA without cleavage when nuclease activity is disabled (paragraph 0551, 0577), corresponding to a site-specific promoter activation complex.
Hou does not expressly teach that the inserted donor polynucleotide comprises a non-functional promoter operably linked to the coding sequence, wherein the coding sequence is initially not expressed, and then is activated by a site-specific promoter activation complex binding upstream of the inserted donor polynucleotide.
Kloti teaches a modified minimal promoter operably linked to a downstream structural coding sequence and positioned downstream of one or more Gal4 upstream activating sequences (p13, line 55-68); Kloti teaches that the structural coding sequence “will not be transcribed (i.e. the construct will be silent)” unless the specific Gal4-2xVP16 trans-activator is present (p11, line 37-46). Kloti further teaches that the Gal4 DNA-binding domain binds the upstream activating sequence and that the VP16 activation domain initiatives transcription of the downstream structural coding sequence (p12, line29-32). Thus, Kloti teaches a gene expression cassette in which the coding sequence is not expressed until a sequence-specific transcriptional activation complex binds upstream of the promoter. Kloti further teaches transformation of the silent cassette into plant cells and activation of the cassette in rice and Arabidopsis when the trans-activator is subsequently provided (p11, line 36-59).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the donor insertion method of Hou by incorporating the conditionally activated, silent promoter arrangement taught by Kloti. Kloti teaches maintaining a coding sequence in a transcriptionally silent state until a sequence-specific transcriptional activator binds an upstream activating sequence, thereby permitting controlled expression of the coding sequence.
A person of ordinary skill in the art would therefore have had reason to incorporate Kloti’s silent promoter cassette into Hou’s donor polynucleotide because Hou teaches targeted insertion of donor sequences comprising selectable markers or agronomic trait sequences and recovery of cells containing the desired insertion, while Kloti teaches a known plant expression arrangement in which an inserted coding sequence remains unexpressed until an upstream-bound site-specific transcriptional activator initiates transcription.
The combined teachings would therefore have predictably resulted in inserting a donor polynucleotide comprising a silent promoter-coding-sequence cassette into a genomic target site, subsequently binding a site-specific promoter activation complex upstream of the inserted cassette, expressing the coding sequence following activation, and selecting and obtaining the target cell expressing the coding sequence.
According, Claim 1 is prima facie obvious over Hou in view of Kloti.
Claim 2 recites the method of claim 1, wherein the coding sequence comprises an agronomic trait, or any combination thereof. Claim 3 recites the method of claim 1, wherein the coding sequence comprises a selectable marker. Claim 4 recites the method of claim 3, wherein the selectable marker is selected from the group of nptII, pat, bar, dsm-2, ahas, gox, gat, gus, a fluorescent protein, or any combination thereof. Claim 5 recites the method of claim 1, wherein the coding sequence confers resistance to an herbicide or an antibiotic.
Hou teaches a “polynucleotide of interest” includes any nucleotide sequence encoding a protein or polypeptide that improves desirability of crops, including herbicide-resistance, insecticidal resistance, disease resistance.(paragraph 0502).
Hou teaches “DNA segments that encode products which can be readily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP)…”(paragraph 0608).
Hou teaches “selectable markers include genes that confer resistance to herbicidal compounds”(paragraph 0609); “DNA segments that encode products which provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin…” (paragraph 0608).
Claims 2-5 are prima facie obvious over Hou in view of Kloti.
Claim 6 recites the method of claim 1, wherein the non-functional promoter comprises a minimal promoter. Claim 7 recites the method of claim 6, where in the minimal promoter comprises a TATA box, a CAAT box, a transcription starting site, an RNA polymerase binding site, or any combination thereof.
Kloti teaches a modified “minimal promoter” that is positioned downstream of an upstream activating sequence and upstream of a structural coding sequence (p13, line 55-68). Kloti teaches that the modified minimal promoter-containing cassette remains silent until the corresponding trans-activator is present (p11, line 37-46). Kloti further describes the minimal promoter as the minimal element necessary for transcription and teaches its use in a chimeric gene together with an upstream activating sequence and downstream structural coding sequence (p11, line15-59).
It would have been obvious to employ Kloti’s modified minimal promoter as the non-functional promoter in the donor cassette of Hou because Kloti expressly teaches that such a promoter arrangement remains transcriptionally silent in the absence of the upstream-binding trans-activator and becomes transcriptionally active when the activator is supplied.
Claims 6-7 are prima facie obvious over Hou in view of Kloti.
Claim 14 recites the method of claim 1, wherein the site specific-promoter activation complex is bound within 1 - 10,000 bp of the non-functional promoter.
Kloti teaches placement of Gal4 upstream activating sequence upstream of and functionally associated with the modified minimal promoter, such that binding of Gal4-2xVP16 to the upstream activating sequence activates the downstream promoter and coding sequence.
It would have been obvious to position the site-specific promoter activation complex within the claimed range of 1-10,000 base pairs from the non-functional promoter because Kloti expressly teaches that the upstream activating sequence is positioned sufficiently close to the modified minimal promoter to permit activation of transcription. The claimed distance encompasses Kloti’s disclosed promoter arrangement, and selecting a particular binding location within this broad range would have been no more than routine optimization.
Accordingly, Claim 14 is prima facie obvious over Hou in view of Kloti.
Claim 18 recites the method of claim 1, wherein the cell is a plant cell. Claim 19 recites the method of claim 18, wherein the plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.
Hou teaches plant cells as target cells for genome editing (claim 14). Hou further teaches the plant cell is a monocot cell or a dicot cell (claim 15). Kloti additionally teaches use of its silent promoter and transactivation system in rice and Arabidopsis plant cells.
Accordingly, claims 18 and 19 are prima facie obvious over Hou in view of Kloti.
Claims 9-12, and 15-17 are rejected under 35 U.S.C. §103 as being unpatentable over Hou (US20200190494A1) in view of Kloti (US6331663B1) as apply to claim 1, and Lowder (Levi G. Lowder et. al., Molecular Plant (2018) 11, pp245–256).
Claim 1 as the teachings of Hou and Kloti are discussed above.
Claims 9, 15-17 are interpreted as dependent of claim 1.
Claim 9 recites the method of claim 1, wherein the site specific-promoter activation complex comprises a site-specific binding protein operably linked to at least one activation domain. Claim 10 recites the method of claim 9, wherein the site-specific binding protein is a CRISPR protein, zinc finger protein, or a TALEN protein. Claim 11 recites the method of claim 10, wherein the CRISPR protein is mutagenized to inactivate nuclease activity. Claim 12 recites the method of claim 9, wherein the activation domain is RTA, TFIIA, TFIIB,TFIID, TFIIE, TFIIF, TFIIH, VP64, VP16, VP160, GAL4, EDLL, ERF2, CBF1, ORCA2,DREB 1A, LEAFY, or any combination thereof.
For the same reason set forth above with respect to claim 1, Hou teaches a Case endonuclease fusion protein comprising heterologous protein domains with transcription activation activity, including VP16 or VP64 (paragraph 0551), Hou teaches CRISPR/Case systems (paragraph 0542-0544).
Lowder teaches a programmable CRISPR transcriptional activation system comprising nuclease-deactivated Cas9 fused to the VP64 transcriptional activation domain (p245, right column, “RESULT”). Lowder teaches that guide RNA directs the dCas9-VP64 complex to a selected promoter region, where recruitment of the VP64 activation domain activates transcription of the target gene (Abstract). Lowder further demonstrates dCas9-VP64-mediated transcriptional activation in plants, including Arabidopsis and rice (p251, “DISCUSSION”).
Accordingly Claim 14 is prima facie obvious over Hou in view of Kloti and Lowder.
Claim 15 recites the method of claim1, wherein the coding sequence is expressed at a higher level that is 1% - 100% higher than the initial low-level expression of the coding sequence. Claim 16 recites the method of claim1, wherein the site specific-promoter activation complex drives expression of the coding sequence by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher than a coding sequence that is driven by only the non-functional promoter. Claim 17 recites the method of claim 1, wherein multiple copies of the site-specific promoter activation complex are bound to the genomic region upstream of the inserted donor polynucleotide such that the site-specific promoter activation complex functions to drive the non-functional promoter.
Hou teaches increases in characteristics inducing increases of at least 1% to at least 400% or more higher than the control (paragraph 0506). Lowder teaches that dCas9-VP64 activated target genes in Arabidopsis, including approximately 7-fold activation of PAP1 and up to approximately 200-fuld activation of FIS2 (p247, right column, pa2). Lowder further teaches that recruitment of additional VP64 activation domains using modified guide-RNA scaffolds produce approximately 30- to 45-fold activation of PAP1 and up to approximated 1,5000-fold activation of FIS2 (p249, left column, pa1). Lowder also teaches multiplexing multiple guide RNAs directed to promoter regions to obtain enhanced transcriptional activation (p246, left column, pa3).
Thus, Lowder teaches expression increases encompassing the recited fold-increase ranges and taches the use of multiple promoter-targeted activation components to enhance expression. Selection of a particular activation level within the claimed ranges would have been a result-effective-variable optimization based on the number and location of guide RNAs and activation domains.
Accordingly, claims 15-17 are prima facie obvious Hou in view of Kloti and Lowder.
Response to Applicant’s Remarks:
1)Applicant argues that Hou and Abbitt do not teach or suggest the claimed activation-dependent selection workflow in which a donor polynucleotide is inserted with a non-functional promoter, followed by activation of the inserted coding sequence and selection of the expressing target cell.
Applicant’s arguments have been fully considered but art not persuasive. The rejection has been modified to rely on Hou in view of Kloti and Lowder rather than Hou in view of Abbitt.
To the extent Applicant argues that the claimed method requires a specific sequence of steps in which the donor polynucleotide is first inserted with a non-functional promoter, followed by activation of the inserted coding sequence and then selection of an expressing target cell, the argument is not persuasive. The claims do not expressly recite such an order of performance, and neither the claim language nor the specification requires the recited steps to be performed in the particular sequence. See MPEP 2111.01; ” Altiris Inc. v. Symantec Corp., 318 F.3d 1363, 1371 (Fed. Cir. 2003).
Hou teaches targeted insertion of a donor polynucleotide comprising a selectable marker or agronomic trait coding sequence, obtaining cells containing the inserted donor sequence, and use of Cas fusion proteins comprising transcriptional activation domains such as VP16 or VP64. Kloti expressly teaches a modified minimal promoter cassette in which the downstream coding sequence “will not be transcribed”, and the construct remains silent unless a sequence-specific Gal4-2xVP16 trans-activator is present. Thus, Kloti teaches a promoter configuration in which expression of the downstream coding sequence is dependent upon the presence of a transcriptional activator. Lowder teaches a programmable CRISPR transcriptional activation system comprising nuclease-deactivated Cas9 fused to VP64 and direct by guide RNA to selected promoter regions in plants. Lowder therefore teaches a site-specific promoter activation complex capable of activating transcription from a selected promoter without cleaving the target DNA.
Accordingly, the combined teachings of Hou, Kloti, and Lowder provide the claimed donor insertion and transcriptional activation feature. Applicant’s asserted sequence of inserting the donor, subsequently activating the coding sequence, and thereafter selecting an expressing cell is not a limitation required by the claims and therefore does not distinguish the claimed subject matter from the combination of references.
2)Applicant further argues that neither Hou nor Abbitt recognizes using a conditionally activated donor cassette as a selection mechanism.
This argument is not persuasive because obviousness does not require that a single reference expressly describe the claimed invention or recognize the same purpose identified by applicant. The combined teachings would have resulted in inserting a silent selectable cassette, activating expressing after insertion, and selecting and obtaining cells expressing the inserted selectable coding sequence. Hour already teaches selectable coding sequences and recovery of cells containing the desired insertion. Kloti and Lowder provide the known silent-promoter and programmable-activation features need to perform that selection after activation.
Conclusion
No claims are allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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