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 .
DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.
Applicant's submission filed on 9/03/2025 has been entered. Applicant indicates on the Request for Continued Examination (RCE) Transmittal that the response of 9/03/2025 be considered.
Claim status
Applicant has amended Claims 1, 36, and 40, and added new Claim 96.
Claims 2-4, 11, 25, 49-50, 65, and 95-96 are pending but withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim.
Claims 1, 17, 20, 22, 28, 36 and 40 are under examination
Election/Restrictions
Applicant’s election of the following invention without traverse in the reply filed on 12/23/2024 has been previously acknowledged.
Group I, claims 1-4, 11, 17, 20, 22, 28, 36, 40, 49, and 95-96, drawn to a polynucleotide composition encoding DNMT3, dCas9 and KRAB.
Claims 25, 50 and 65 have been withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable linking claim.
Applicant’s election of the following species has been previously acknowledged.
Applicant elected the construct of Formula IIIb-1:
5’-(A-B)-CasN-CasC-Tp-Kr-3’, wherein
A and B are DNMT3A and DNMT3L,
CasN and CasC are N-term and C-term of dCas9,
K is KRAB,
T is an antibody epitope, self-cleaving peptide, and the antibody,
p is 1, r is 1; wherein
DNMT3A comprises SEQ ID NO:69,
DNMT3L comprises SEQ ID NO:74,
dCas9 comprises SEQ ID NO:1,
KRAB comprises SEQ ID NO:53,
GCN4 is the epitope for binding an scFv.
Claims 2-4, 11, 25, 49 and 95-96 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic claim.
Note that newly submitted claim 96 directed to species of SEQ ID NOs that are independent or distinct from the species originally elected. Since applicant has received actions on the merits for the originally presented species, this species has been constructively elected by original presentation for prosecution on the merits. Accordingly, new claim 96 is withdrawn from consideration as being directed to a non-elected species. See 37 CFR 1.142(b) and MPEP § 821.03.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 9/15/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Declaration under 37 CFR 1.132
The declaration under 37 CFR 1.132 filed by Dr. Shaoshuai Mao on 9/03/2025 is considered, but insufficient to overcome the rejection of instant claims based upon 35 U.S.C 103 for the reasons set forth below.
Withdrawn 35 USC § 103
The prior rejection of Claims 1, 17, 20, 22, 28, 36 and 40 under 35 U.S.C. 103 as being unpatentable over Maeder et al. (US2024/0076678, filed 6/20/2023, CON of PCT/US2021/064913, filed 12/22/2021, prior art of record), in view of in Tanenbaum et al. (US2017/0219596, filed 7/14/2015, prior art of record), is withdrawn in order to incorporate the prior art of Yamagata et al. (US2021/0260170, filed 5/17/2018) and Morita (Nat Biotech, 2016,34(1):1060-1070).
New 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 of this title, 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 17, 20, 22, 28, 36 and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Maeder et al. (US2024/0076678, filed 6/20/2023, CON of PCT/US2021/064913, filed 12/22/2021, prior art of record), in view of in Tanenbaum et al. (US2017/0219596, filed 7/14/2015, prior art of record), Yamagata et al. (US2021/0260170, filed 5/17/2018) and Morita (Nat Biotech, 2016,34(1):1060-1070).
In regard to claim 1, Maeder teaches a construct comprising a methyltransferase domain, a dead Cas9 DNA binding domain, and a repressor domain ([0016, 0400, 0589], see claim 135 of Maeder). In one example Maeder teaches a construct of the following formula ([0011, 0039, 0511], Example 7, see Fig. 6B excerpt below):
PNG
media_image1.png
113
1221
media_image1.png
Greyscale
Specifically in regard to the claimed Formula IIIb-1, the construct of Maeder comprises from 5’ to 3’ an A and B as DNMT3A and DNMT3L, CasN and CasC as dSpCas9, T as the linker XTEN16, and K as KRAB, thereby generating the formula 5’-(A-B)-CasN-CasC-T1-K1-D0-3’.
However, although Maeder teaches the linker region may comprise cleavable peptide (e.g., a 2A peptide) [0381], and Maeder teaches the linker region may comprise a Sun Tag-like peptide array that comprises multiple copies of an epitope tag that can link multiple effector domains attached to antibody sequences recognizing the epitope tag, wherein the peptide array comprises GCN4 epitopes [0384], they are silent with respect to a “T” linker after the dCas9 and before the transcriptional repressor KRAB that comprises a Sun Tag peptide array of GCN4 epitopes, followed by a cleavable T2A peptide, and then epitope specific antibodies that are fused to the KRAB effector domain.
Nevertheless, Maeder cites the prior art of Tenenbaum et al. (US2017/0219596) [0384].
Tanenbaum teaches a dCas9-Sun Tag with a linker comprising a peptide array of GCN4 epitopes (e.g., SEQ ID NO:1 or 2) that bind an anti-GCN4 scFv fused to a transcriptional regulatory domain ([0042-0043, 0083, 0085 0090-0092], see Fig. 8). Tanenbaum teaches that the transcriptional regulatory domain can be a transcriptional repressor domain such as KRAB [0092].
Accordingly, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to prepare the DNMT3A/L-dCas9-XTEN16-KRAB construct as taught by Maeder, and substitute the XTEN16 linker with a Sun Tag based GCN4 epitope array, a 2A cleavable linker, and anti-GCN4 scFv attached to the KRAB effector domain as suggested by Maeder in view of Tanenbaum with a reasonable expectation of success. The ordinary skilled artisan would have been motivated to do so for several reasons. First, as stated supra, Maeder explicitly suggests incorporation of the GCN4 peptide array and cites the prior art of Tanenbaum as an enabling disclosure, thus it would have been predictably obvious to have substituted the single fusion protein of Maeder for the two fusion proteins of Tanenbaum. Furthermore, there were known advantages to the Sun Tag system including Tanenbaum’s general teachings that there would have been a clear benefit to substituting a linker that joins a single transcriptional regulatory domain with a linker that recruits many transcriptional [AltContent: textbox ([img-media_image2.png])]regulatory domains to potently control gene transcription ([0043], see also modified Fig. 8 of Tanenbaum adjacent). Moreover, the related art of Yamagata et al. (2021) teaches that multiple repressors are recruited to the dCas9 protein via the Suntag system [0031]. Specifically, Yamagata teaches that when the DNA encoding the dCas9-Suntag and the DNA encoding anti-GCN4 antibody-transcription repressor are coexpressed within a host cell, a multiple transcriptional repressor-CRISPR effector protein complex can be formed in the cell [0042].
Furthermore, in regard to the inclusion of the cleavable 2A peptide linker as suggested by Maeder, the related art of Morita et al. (2016) teaches a cleavable 2A peptide linker to make an “all-in-one” vector comprising a dCas9-SunTag and anti-GCN4 antibody-transcriptional regulator (Methods, 3rd para.). Thus, it would have been obvious to include the 2A cleavable peptide so as to ensure that the DNMT3-dCas9 and the scFv-KRAB would be expressed in the same cell.
In regard to claim 17, Maeder teaches the human DNMT3A domain comprises the amino acid sequence of SEQ ID NO: 34 ([0006, 0011, 0014, 0018, 0036], see p. 113, Sequence Table, see Claims 143-144 of Maeder), which is 100% identical to the claimed SEQ ID NO:69 with the exception of the start methionine (see SCORE search 1/28/2025, rag.file). Nevertheless, this would have been a necessary and obvious element for the initiation of translation of the taught construct.
In regard to claim 20, Maeder teaches the human DNMT3L domain comprises the amino acid sequence of SEQ ID NO: 37 (p. 114, Sequence Table, see Claims 143-144 of Maeder), which is 100% identical to the claimed SEQ ID NO:74 (see SCORE search 1/28/2025, rag.file, Result No. 1).
In regard to claim 22, Maeder teaches the KRAB domain is from ZIM3, and comprises the amino acid sequence of SEQ ID NO: 67 ([0012-0013, 0815], p. 62, Table 2, line 1, p. 119, Sequence Table, see Claim 136 of Maeder), which is 100% identical to the claimed SEQ ID NO:53 (see SCORE search 1/28/2025, rag.file, Result No. 1).
In regard to claim 28, Maeder teaches the dSpCas9 comprises the amino acid sequence of SEQ ID NO: 3 ([0010], p. 103, Sequence Table), which is 100% identical to the claimed SEQ ID NO:1(see SCORE search 1/28/2025, rag.file).
In regard to claims 36 and 40, as stated supra, Tanenbaum, Yamagata, and Morita teach at least one GCN4 epitope in the Sun Tag linker, which binds to at least one anti-GCN4 scFv connected to the transcriptional regulator.
Hence, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
RESPONSE TO ARGUMENTS
Applicant's arguments filed on 9/03/2025, and the declaration by Dr. Shaoshuai Mao are acknowledged.
First, Applicant’s argue and Dr. Mao declares that the incorporation of the Suntag system allows a single DNMT-dCas9-nxGCN4 unit to recruit multiple scFv-KRAB molecules in vivo, thereby amplifying the repressive effect. Moreover, the two fusion proteins are markedly smaller than the single DNMT-dCas9-KRAB fusion protein, thereby minimizing steric interference among the individual components during folding, allowing each functional domain to remain more accessible so as to achieve more potent transcriptional repression compared to the singe fusion protein. Moreover, the n-xGCN4 tag forms high density KRAB effector clusters to suppress the target gene, while the methyltransferase DNMT3A/L enable long-term suppression. Specifically, Applicant argues and Dr. Mao declares that the claimed system results in sustained long-term epigenetic silencing that remains evident 7 days post-transfection (Fig. 2E), and from days 9-23 the percentage of silenced cells without sorting declined modestly from 19% to 13%.
Second, Applicant’s argue and Dr. Mao declares that the two DNMT-dCas9-1xGCN4 and scFv-KRAB fusion proteins are unequivocally more effective at [AltContent: textbox ([img-media_image3.png])]suppressing the PCSK9 gene compared to the single DNMT-dCas9-KRAB fusion protein. Specifically, Dr. Mao presents in vivo data measuring plasma PCSK9 levels between DNMT-dCas9-1xGCN4 and scFv-KRAB (V10) and DNMT-dCas9-KRAB (V11) treated mice (see excerpt adjacent), as evidence of unexpected results from the claimed construct.
Finally, Applicant argues and Dr. Mao declares that Example 1 (Figs. 2C-2E) of the specification demonstrates that the DNMT3-dCas9-10xGCN4-T2A-scFv-KRAB significantly and unexpectedly repressed transcription compared to the conventional DNMT3-dCas9-KRAB control, wherein target gene suppression is enhanced by over 30% and sustained exceeding 23 days. Applicant compares this to Maeder’s results, wherein only 10% inhibition is achieved that dissipates within 9 days.
Applicant's arguments and Dr. Mao declaration have been fully considered but they are not persuasive.
In regard to Applicant’s and Dr. Mao’s first arguments directed to the advantages of the claimed system comprising both the methyltransferases DNMT3A/L and KRAB repressor domains in general, as stated in the pending rejection Maeder et al. teach the same components and would therefore be expected to exhibit the same advantages. Although Maeder does not directly compare the DNMT3 only or KRAB only systems, it was well known that the combination of a methyltransferase domain and KRAB repressor domain led to prolonged gene suppression compared to either domain alone. For example, the prior art of Gilbert (WO2022/140577, filed 12/22/2021, see IDS filed 3/13/2025) teaches a construct similar to Maeder comprising a DNMT3A/L-dCas9-KRAB fusion (see Fig. 1A, “CRISPRoff-V2” of Gilbert), and demonstrates that the combined fusion protein silences gene expression (i.e., GFP reporter) nearly twice as well as either dCas9-KRAB or dCas9-DNMT3A/L alone, with prolonged suppression to above 80% for over 50 days (see Figs. 1C & E of Gilbert). Furthermore, in regard to the advantages of the Sun tag system, as stated in the pending rejection, both Tanenbaum and Yamagata teach the advantage that a single DNMT-dCas9-nxGCN4 unit can recruit multiple scFv-KRAB molecules in vivo, thereby amplifying the repressive effect. In regard to the folding advantage of two smaller fusion proteins compared to a single fusion protein, Tanenbaum teaches the advantage of the Sun tag is that epitopes can be inserted into a region of fusion protein that is solvent exposed when the protein is in a folded conformation [0078], and that the spacing of these epitopes (i.e., GCN4 peptides) are optimized to limit steric hindrance between the Sun tagged fusion protein and scFV-effector molecule [0140], thereby allowing each functional domain to remain more accessible so as to achieve more potent transcriptional repression compared to the singe fusion protein.
In regard to Applicant’s and Dr. Mao’s second arguments directed to the purported unexpected results exhibited by the claimed construct, MPEP 716.02(b) states that to be of probative value, any objective evidence should establish “that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance.” In instant case, the figure included in the Dr. Mao’s declaration does not appear to show a statistically significant difference between the DNMT-dCas9-1xGCN4 and scFv-KRAB (V10) and DNMT-dCas9-KRAB (V11) treated mice as the error bars are overlapping. Nevertheless, if Applicant was able to furnish to the examiner that this was a statistically significant difference, the effects of a 1xGNC4 tag yielding a statistically significant difference in repression over the single fusion protein would be worthy of secondary considerations as the use of a single GNC4 tag would not recruit many KRAB domains, but a single KRAB domain. Furthermore, the examiner recommends to Applicant to amend instant claims to a 1x-GCN4 so as to ensure that the objective evidence of nonobviousness is commensurate in scope with the claims. See MPEP § 716.
In response to arguments directed to the purported unexpected results presented in the originally filed disclosure, although Applicant’s specification indicates that the “Control” is indeed the DNMT3-dCas9-KRAB fusion protein of Fig.1 (p. 10, Detailed Description of Fig. 2), this fusion protein is paired with the “Nt sgRNA”, which appears to be a negative control (see p. 193, Table 4 of Applicant’s disclosure). Thus, the Examiner cannot reasonably compare the Sun Tag systems to the “Control” of Fig. 2. Furthermore, contrary to Applicant’s assertion and Dr. Mao’s declaration, Fig. 1 demonstrates that the single DNMT3-dCas9-Krab fusion can repress CD81/151+ cells to about 0.6% on Day 7, while Fig. 2E demonstrates that the two part DNMT3-dCas9 + scFv-Krab fusions repress CD81/151+ cells to about 0.75% on Day 7. Thus, there does not appear to be any unexpected result according to Applicant’s specification, and in fact, the Sun Tag system appears to repress less than the single fusion protein. Applicant is reminded that arguments of counsel cannot take the place of factually supported objective evidence in the record. See In re Schulze, 346 F.2d 500, 602, 145 USPQ 716, 718 (CCPA 1965), In re Huang, 100 F.3d 135, 139-40, 40 USPQ2d 1685, 1689 (Fed. Cir. 1996); In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984).
Moreover, as stated supra, it was well known that the 10x-GCN4 Sun tag system typically exhibited superior results compared to a single fusion protein. For example, Tanenbaum uses the 10xGCN4 Sun Tag for linking multiple copies the activation domain of VP64 (see Fig. 8A), and they demonstrate the scientific principle that recruiting multiple copies of a transcriptional regulator is much more effective than a single fused copy (see Fig. 8C, wherein the Sun Tag increase gene regulation by 10-50 fold). Similarly, Morita demonstrates that compared to the direct fusion of the dCas9 to a TET1 demethylase (i.e., “System 1”), the SunTag version (i.e., “System 3”) is approximately 3 fold more effective at gene regulation (Figs. 1a & 1e). Thus, the results presented by Applicant’s Example 1 are not necessarily surprising and in fact confirm that the invention works as predicted by the prior art. This is not relevant to the issue of nonobviousness of the claimed subject matter and provides no objective evidence thereof. See MPEP § 716
In view of the foregoing, when all of the evidence is considered, the totality of the rebuttal evidence of nonobviousness fails to outweigh the evidence of obviousness.
Conclusion
No claims are allowed.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ARTHUR S LEONARD whose telephone number is (571)270-3073. The examiner can normally be reached on Mon-Fri 9am-5pm.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, James Doug Schultz can be reached on 571-272-0763. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ARTHUR S LEONARD/ Examiner, Art Unit 1631