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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 11/26/2025 has been entered.
Claims 1-4 are pending. Applicant amended claim 1 by amendment filed on 11/26/2025. Claims 3-4 are withdrawn from consideration pursuant to 37 CPR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claims.
Therefore, claims 1-2 are currently under examination to which the following grounds of rejection are applicable.
Priority
The instant application is a national stage entry under 35 USC 371 of PCT/JP2020/015291 filed on 04/03/2020, which claims benefit of foreign filed application JP2019-072782 filed on 04/05/2019.
In the non-final rejection filed 12/03/2024, the earliest possible priority was determined as 04/05/2019 due to the claim of foreign benefit to JP2019-072782.
Filing of a certified untranslated copy of the Japanese application JP2019-072782, filed October 10, 04, 2021 was previously acknowledged.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Withdrawn Rejections in response to Applicants’ arguments or amendments
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1-2 under 35 USC § 112(b) is withdrawn in view of the amendments in the response filed on 11/26/2025.
Applicant’s arguments with regard to a withdrawn objection/rejection are moot.
Maintained and modified rejections in response to Applicants’ arguments or amendments
Claim Interpretation in view of claim amendments
Regarding the amendments to claims as set forth in in the response filed on 11/26/2025:
Applicant asserts on Pg. 7 of the remarks filed 11/26/2025 that it is clear from the amended claim 1 that the first and the second nucleases target and cleave different sequences recited in the claims. This is illustrated in Figures 1, 2A, 3A and 4A of the specification as filed. Thus, nucleases are not limited to those that depend on sgRNA. This is described, for example, in paragraph spanning pages 11 and 12 of the specification as filed.
Although examiner agrees that claim 1 is not limited to only nucleases that depend upon sgRNA, under the broadest reasonable interpretation nucleases that depend upon sgRNA cannot be excluded. Therefore, nucleases that depend upon sgRNA will still read upon the nucleases recited in the claim 1. Further, the applicants disclosure provides that the nuclease system made up of two nucleases can be a CRISPR/CAS system (please see claim 2). Therefore, the recited nucleases can be interpreted as both being CRISPR guided by multiple distinct guide RNA.
Claim Rejections - 35 USC § 103
Claim(s) 1-2 remain rejected under 35 U.S.C. 103 as being unpatentable over Feng et al. (US 20170198302 A1, 2017, of record) in view of Gratz et. al (Genetics vol. 194,4 (2013): 1029-35, of record).
Regarding Claims 1-2, Feng teaches a use of a site-specific nuclease system (CRISPR/Cas9) (for claim 2) and a donor sequence encoding for a 5’ side homology arm, a donor sequence, and a 3’ side homology arm genome homologous segments being homologous to a predetermined genome sequence (Pg. 4, Paragraph 0031).
Moreover, Feng teaches introducing to a cell a donor sequence, a CAS9, an sgRNA (sg-2 or sg-3) that targets a sequence in the 5’ homologous arm of the donor construct (GAPDH donor-HDR.2) and a sgRNA (sg-4) that targets a sequence in the 3’ homologous arm of the donor construct (Pg. 9, Paragraph 0085 and Pg. 26, Paragraphs 0229-0232 and Fig. 9) (for claims 1-2).
An sgRNA hybridizes to a specific nucleotide sequence, and the donor homology arm contains sequence homologous to the targeted genomic region, the CRISPR/CAS9 complex necessarily cleaves both the donor homology arm sequence and the homologues genomic sequence. Therefore, Feng teaches a nuclease system that targets and cleaves sequences (i) and (ii) of instant claim 1.
Although Feng teaches that these sgRNA can hybridize to the donor construct and the segment within the upstream or downstream non- coding sequence of the predetermined genome sequence, Feng does not specifically teach a simultaneous targeting of both a sequence on the 3’ side downstream of the site targeted by sg-2 or 3 on the genome, and targeting a sequence on the 5’ side upstream of the site targeted by sg-4 on the genome.
Gratz teaches that by combining two specific guide RNAs, termed ChiRNA, that it is possible to design one ChiRNA to recognize and bind the 5’ prime end of a target gene with a second ChiRNA designed to recognize and bind the 3’ prime end of a target gene (Figure. 1.) These ChiRNA’s were successfully used with a CRISPR/Cas9 nuclease system in drosophila to target and cleave at both a 5’ side upstream and 3’ location downstream of the genome target region for the introduction of a donor sequence harboring the attP gene to successfully replace the gene yellow. introduce the donor sequence attP to replace the yellow gene (Figure. 1G-H, and Section: Cas9-mediated homologous recombination).
Additionally, Gratz teaches that combinatorial usage of this CRISPR/CAS9 system with the multiplex targeting capabilities of a precisely designed plurality of guide RNAs is an effective method (Pg. 1030, Paragraph 6) and reduces off-target cleavage (Pg. 1033, Paragraph 2). Moreover, Gratz teaches the only limiting requirements of their targeting system is 20 nucleotides (nt) of homology between the chiRNA and its genomic target. Cleavage also requires that the 3’ end of the genomic target sequence contains a 3-basepair (bp) proto-spacer adjacent motif (PAM) sequence, NGG, Thus, selection of a 20-nt target sequence is limited only by the requirement for an adjacent PAM sequence (Pg. 1030, Results, 1st Paragraph). Therefore, Gratz teaches cleavages that would encompass a second genomic cleavage (iii of the instant claim 1) positioned downstream (or upstream in the mirror configuration) relative to the first genomic cleavage sites. The method of Gratz and the respective ChiRNA design is inherently modular and defined only by simple constraints.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of application to modify the method of Feng which teaches CRISPR/Cas9 mediated donor insertion to produce a genetically modified cell using a site-specific nuclease system to further include within the method the multiplex, site-specific guide RNA strategy taught by Gratz. One would have been motivated to combine these teachings as the inclusion of the method of Gratz would enable simultaneous cleavage at flanking sites located 5’ side upstream and 3’ side downstream of sequences targeted by the sgRNAs taught by Feng.
There would have been reasonable expectations of success in combining these teachings as one of ordinary skill in the art would recognize to combine known elements in the art to give predictable results.
Response to Applicants’ Arguments as they apply to the rejection of claim(s) 1-2 under 35 U.S.C. 103
At pages 7-11 of the remarks filed on 11/26/2025, Applicants essentially argue that the rejection of claims be withdrawn as “…the cited references do not teach or suggest a method where both NHEJ and HR are used for inserting a donor sequence into a genome and do not provide the required reasonable expectation that such a combination can be made successfully.”
Applicants’ arguments have been considered, but have not been found persuasive.
On page 8 of the remarks, Applicant argues,
“…that claim 1 recites that sequences (i) and (ii) of Groups A and B are targeted and cleaved by the first nuclease of the site-specific nuclease system. Thus, the donor DNA (which contains sequence (i), i.e., the homology arm sequence) and the genome-editing target region on a genomic DNA (which contains sequence (ii), i.e., the nucleotide sequence of the genome-editing target region homologous to the respective homology arm) can only be joined together by NHEJ at the cleavage site. On the other hand, because the donor sequence contains a second homology arm sequence (which is not cleaved by a nuclease) and the genome-editing target region contains a sequence homologous to said second homology arm (also not cleaved by the nuclease), integration by HR will occur at that site while NHEJ will occur.”
Claim 1 does not recite a specific repair pathway. Claim 1 requires only cleavage of defined sequences (i), (ii), and (iii), and introduction of donor DNA. The mechanism by which the cell would repair is not recited as a claim limitation. Furthermore, absence clear evidence to the contrary, cleavage of sequences (i) and (ii) does not dictate that repair must occur exclusively through NHEJ without any possibility of repair through HDR. A double stranded break (DSB) in the presence of homologous donor DNA can be repaired through HDR.
At Pg. 10, applicant asserts,
“Contrary to the Examiner assertion, Feng does teach a simultaneous use of sg-3 that targets a sequence in the 5' homology arm of the donor construct and sg-4 that targets a sequence in the 3' homology arm of the donor construct and shows the result that the simultaneous use induced NHEJ-targeting on both sides (Please see the last sentence of paragraph [0231] of Feng). This fact means that even if one of ordinary skill in the art prior to the effective filing date of the present application had modified the method of Feng to combine (i) an sgRNA that targets a sequence in the 5' homology arm of the donor construct, and (ii) an sgRNA that targets a sequence in the 3' homology arm of the donor construct for the multiplex targeting taught by Gratz, they would not have been able to induce knock-in via both NHEJ and HDR as recited in the present claims.”
As discussed above, the claims do not recite any repair mechanism or require induction of both NHEJ and HDR. The claims only require cleavage of defined sequences and introduction of donor DNA. Repair pathway selection is an inherent cellular process and is not recited as a limitation of the claims.
Accordingly, applicants’ argument is not commensurate in scope with the claims.
At pages 10-11, applicant asserts, “In the presently claimed method, knock-in via both NHEJ and HDR can be induced by using a combination of (i) the first nuclease that targets both genomic DNA and donor DNA, and (ii) the second nuclease that targets only genomic DNA The knock-in efficiency of the presently claimed method is as high as 16.6% to 33.3% in vivo (Please see "Combi" of Table 2 on page 43), while that of Feng is only about 8% in vitro when the combination of sg-3 and sg-4 is used (Please see "sg-3&sg-4" of Fig. 9B). Neither Feng nor Gratz teaches or suggests an effect by inducing knock-in via both NHEJ and HDR. Thus, the claimed method provides an unexpected
advantageous result. Therefore, the cited references do not teach or suggest a method where both NHEJ and HR are used for inserting a donor sequence into a genome and do not provide the required reasonable expectation that such a combination can be made successfully.”
The claims are not limited to the specific experimental conditions relied upon by the applicants’ argument. The improvement in efficiency has not been shown to be commensurate in the scope with the claims nor attributable to a specific feature that is not taught by the prior art.
Accordingly, the asserted efficiency increase does not overcome the prima facie case of obviousness.
Conclusion
Claims 1-2 remain rejected. No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KODYE LEE ABBOTT whose telephone number is (703)756-1111. The examiner can normally be reached M-F 8-5.
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, Maria G. Leavitt can be reached on (571) 272-1085. 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.
/KODYE LEE ABBOTT/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634