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 .
Application Status
This action is written in response to applicant’s correspondence received 27 October 2025. Claims 125-126 and 249-265 are currently pending. Claims 125-126 are amended. Claims 133, 148-149, 153, 162, 164-165, 168, 174, 181-182, 185, 187-188, 193-194, 203, and 206-207 have been canceled. Claims 249-265 are newly added. Accordingly, claims 125-126 and 249-265 are examined herein. The restriction requirement mailed 26 February 2025 is still deemed proper. Applicant's elected Group I, claims 125-126 and 133, without traverse in the reply filed 21 May 2025.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant' s amendments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Objections
Claim 257 objected to because of the following informalities: the claim recites two instances of “AcrIIA5” in part d) of the claim. Examiner suggests amending the claim to delete the repeated phrase. Appropriate correction is required.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
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.
Claim(s) 125-126, 249, 253, 255-256, 259, 263-264, and 266 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yoon (PG Pub No. WO 2019/123014 A1, filed 21 December 2018) in view of Wei (US Patent No. 11,624,077 B2, filed 8 August 2017).
Regarding claim 125, Yoon is drawn towards an invention concerned with compositions and methods for increasing mutation efficiency and homologous recombination rates of site- specific endonucleases (Abstract). Yoon teaches the use of chimeric polypeptides comprising a 3' to 5' exonuclease fused to a CRISPR Cas enzyme (i.e., an active RNA programmable nuclease) ([00254]). Yoon teaches that two CRISPR Cas9 fusion enzymes, each utilizing their own guide RNAs (i.e., first and second gRNAs), can target two genomic DNA sites such that two double stranded breaks (i.e., breaks that create a lesion between first and second genomic sites) are induced in target genomic DNA followed by exonuclease activity ([00299]; see FIG. 4). Yoon teaches that, subsequently, a single stranded DNA oligonucleotide or a circular dsDNA (i.e., a donor DNA molecule) can be added leading to the intended replacement (i.e., the insertion of the donor molecule into the lesion between the first and second genomic sites) via homology directed repair systems ([00299]; see FIG. 4). Yoon teaches that the circular dsDNA molecule can be linearized via the use of a third fusion protein that utilizes a third guide RNA, sgRNA C, that can induce double stranded breaks on the circular dsDNA ([00442]). Yoon teaches that utilizing the chimeric polypeptides resulted in the efficiency of HR increasing as high as 10-folds ([00299]).
Yoon does not teach or suggest the use of third and fourth gRNA molecules that hybridize to third and fourth fusion proteins in order to direct the fusion proteins towards first and second flanking regions on the donor DNA molecule such that the donor DNA molecule is cleaved prior to the insertion of the donor DNA molecule at the genomic lesion (Claim 125).
However, one of ordinary skill in the art would have considered the teachings of Wei as both references are common fields of endeavor pertaining to the use of insertion of donor DNA sequences at target genomic regions of interest in target cells.
Wei is directed towards an invention concerned with a linear donor construct that can be cleaved in a cell to produce the linear donor DNA (Abstract). Wei teaches the use of a method of knocking-in a donor DNA construct into a genomic region of interest within HeLa cells comprising the use of a linear donor DNA that is flanked on both sides by sgRNA sites termed “sg1” and “sg2” (i.e., third and fourth gRNAs directed towards flanking regions on a donor DNA molecule) (Col. 8, lines 11031; see FIG. 1A-1C). Wei teaches that the donor DNA molecule may be a circular DNA molecule that comprises a linear donor DNA that, when cleaved in a cell, produces linear donor DNA (Col. 12, lines 11-23). Wei teaches that the insertion of the donor DNA at a target genomic region of interest resulted in the successful expression of the donor DNA in the target cells (Col. 8, lines 11031; see FIG. 1A-1C).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the method of knocking in a donor DNA molecule sequence in a target genomic region of interest, as described by Yoon, for a method of utilizing a third and fourth gRNA that targets flanking sequences on the donor DNA molecule and hybridize to third and fourth fusion proteins such that the donor DNA molecule is cleaved prior to the insertion of the donor DNA molecule at the genomic lesion via the substitution of sgRNA C for the third and fourth sgRNAs, as described by Wei. A person of ordinary skill in the art would have had a reasonable expectation of success because Yoon teaches that the circular donor dsDNA molecule can be linearized via the use of a third sgRNA molecule that hydrides to a third fusion protein (i.e., the third sgRNA of Yoon can target the circular donor DNA molecule) while Wei teaches that utilizing a circular dsDNA donor molecule that comprises a linear dsDNA donor molecule can be inserted into a genomic region of interest via the use of two sgRNA molecules that flank a linear donor DNA sequence of interest and direct two Cas9 molecules to cleave the donor DNA molecule such that it can be inserted at a genomic region of interest.
Regarding claim 126, Yoon teaches that a) the two CRISPR Cas9 fusion enzymes, each utilizing their own guide RNAs, can target two genomic DNA sites such that two double stranded breaks are induced in target genomic DNA followed by exonuclease activity ([00299]; see FIG. 4).
Regarding claim 249, Wei teaches that target sequences of Cas9 proteins that utilize an sgRNA to cleavage the target sequence comprise a PAM sequence about 20 nucleotides downstream of the target sequence (i.e., Wei teaches that the first and second flanking region that are targeted and cleaved by Cas9 must comprise a PAM sequence located downstream the first flanking region and a PAM sequence located downstream of the second flanking region in order for the Cas9 to target the donor DNA molecule) (Col. 13, lines 38-51). Yoon further provides support for the PAM location as Yoon teaches that sgRNA C was utilized to guide the Cas9 fusion protein to a target site located before a PAM sequence ([00452]).
Regarding claim 253, Yoon teaches that the cleavages in the genomic sites are double stranded breaks ([00299]; see FIG. 4). Wei teaches that the donor DNA molecule is double stranded and that Cas9 can be directed to flanking regions on the donor DNA in order to cleave the donor DNA (i.e., the cleavages at the flanking regions are double stranded breaks) (Col. 13, lines 38-51).
Regarding claims 255-256, Yoon teaches that the fusion proteins comprise a Cas9 RNA programmable nuclease ([00299]; see FIG. 4).
Regarding claim 259, Yoon teaches that the exonuclease may be a lambda exonuclease ([00114]-[00115]; see FIGs. 83-84).
Regarding claim 263, Yoon teaches that the circular dsDNA is delivered to a cell via a plasmid (i.e., a vector) ([0033]).
Regarding claim 264, Wei teaches that the donor DNA comprises a nucleic acid sequence encoding a functional puromycin gene (Col. 8, lines 11031; see FIG. 1A-1C).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the donor DNA sequence of Yoon for a donor DNA sequence that comprises a functional puromycin gene, as described by Wie. A person of ordinary skill in the art would have been motivated to do so in order to facilitate selective growth conditions of the cells of Yoon such that only cells that were successfully transformed with the donor DNA and fusion proteins could grow. A person of ordinary skill in the art would have had a reasonable expectation of success because both Yoon and Wei teaches the use of dsDNA donor molecules that could be utilized to insert a nucleic acid sequence at a genomic region of interest.
Regarding claim 266, Yoon teaches that i) the first genomic is positioned 5’ to the second genomic site on the endogenous DNA molecule ([00299]; see FIG. 4).
Claim(s) 250-252, 254, and 260-262 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yoon (PG Pub No. WO 2019/123014 A1, filed 21 December 2018) in view of Wei (US Patent No. 11,624,077 B2, filed 8 August 2017) as applied to claims 125-126, 249, 253, 255-256, 259, 263-264, and 266 above, and further in view of Peddle (The Yale journal of biology and medicine 90.4 (2017): 533).
Regarding claims 250-252, 254 and 260-262, Yoon in view of Wei renders obvious claims 125-126, 249, 253, 255-256, 259, 263-264, and 266 as described above. Yoon teaches that the donor DNA has homologous flanking regions to the endogenous DNA molecule ([00299]; see FIG. 4).
Yoon in view of Wei does not teach that the first and second flanking region of the donor DNA comprise a mutated site relative to the endogenous DNA molecule (Claim 25), wherein the mutated site is a mutated PAM (Claim 251), wherein the mutated PAM sites of the first and second flanking regions comprise distinct sequences (Claim 252). Yoon in view of Wei does not teach that a region between the first and second genomic sites is associated with a disease (Claim 254). Yoon in view of Wei does not teach that the first or second genomic site comprises a nucleotide polymorphism (Claim 260) that is allele specific (Claim 261). Yoon in view of Wei does not teach that the homology directed repair treats a disease or disorder (Claim 262).
However, one of ordinary skill in the art would have considered the teachings of Peddle as both references are common fields of endeavor pertaining to the use of donor nucleic acids inserted at genomic regions of interest via Cas9.
Peddle is directed towards a study concerned with the application of CRISPR/Cas9 for the treatment of retinal diseases (Abstract). Peddle teaches that when a double-stranded break occurs in a host cell’s genome the cell will attempt to repair it using one of two processes: NHEJ or HDR (pg. 535). Peddle teaches that supplying a donor DNA molecule containing a desired mutation to a genomic region of interest that was cleaved by a Cas9 molecule allows for the integration of the desired mutation into the target site via HDR (pg. 535). Peddle teaches that Cas9 molecules can be targeted to an allele-specific mutation that is dominant negative and causes Rhodopsin (i.e., a region of a genomic site that is associated with a disease and is allele-specific) such that the mutant allele is disrupted and restoring wildtype functionality to the gene (pg. 536). Peddle further teaches that utilizing novel PAM sites, generated via a mutation, in a dsDNA molecule of interest can ensure that a certain Cas9 only binds to a target strand of the dsDNA molecule comprising the mutated PAM and not to a different nucleic acid (pg. 536). Peddle teaches that many known species of Cas9 and their associated PAM sequences can be utilized to target specific target sequences of interest (pg. 536-537; see Table 2).
Therefore, regarding claims 250-252, 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 DNA molecule rendered obvious by Yoon in view of Wei such that the first and second flanking regions comprised mutated PAMs that were different from one another, as described by Peddle. A person of ordinary skill in the art would have been motivated to do so in order to ensure that the Cas9 associated with each mutated PAM only cleaved the dsDNA donor molecule and not other off-target sequences. A person of ordinary skill in the art would have had a reasonable expectation of success because both Yoon in view of Wei and Peddle teach the use of PAM sites that can be utilized to target Cas9 to nucleic acid sequences of interest in order to effect cleavages at the target nucleic acids.
Therefore, regarding claims 254 and 260-262, 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 method rendered obvious by Yoon in view of Wei such that the region between the first and second genomic sites is associated with a disease and comprises a nucleotide polymorphism that is allele specific and can be restored via the use of a donor DNA molecule, as described by Peddle. A person of ordinary skill in the art would have been motivated to do so in order to treat an allele-specific mutation that is dominant negative and associated with a known disease. A person of ordinary skill in the art would have had a reasonable expectation of success because both Peddle and Yoon in view of Wei teach the use of donor DNA molecules that can be integrated into a target genomic region of interest via the use of Cas9 molecules.
Claim(s) 257-258 and 265 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yoon (PG Pub No. WO 2019/123014 A1, filed 21 December 2018) in view of Wei (US Patent No. 11,624,077 B2, filed 8 August 2017) as applied to claims 125-126, 249, 253, 255-256, 259, 263-264, and 266 above, and further in view of Shin (Science advances 3.7 (2017): e1701620).
Regarding claims 257-258 and 265, Yoon in view of Wei renders obvious claims 125-126, 249, 253, 255-256, 259, 263-264, and 266 as described above.
Yoon in view of Wei does not teach or suggest that the method further comprises delivering a programmable nuclease inhibitor to the target cell (Claim 257) selected from AcrIIA4 (Claim 258). Yoon in view of Wei does not teach or suggest that the donor DNA molecule comprises a nucleic acid encoding an RNA programmable nuclease inhibitor (Claim 265).
However, one of ordinary skill in the art would have considered the teachings of Shin as both references are common fields of endeavor pertaining to the use of Cas9 in genome editing.
Shin is directed towards a study concerned with disabling Cas9 by an anti-CRISPR DNA mimic: AcrIIA4 (Abstract). Shin teaches that the recent and rapid expansion of the Cas9 toolkit for gene editing applications has lacked an inducible off switch to prevent undesired gene editing (pg. 4). Shin teaches that utilizing a plasmid encoding a AcrIIA4 (i.e., a nucleic acid encoding a programmable nuclease inhibitor) and delivering the plasmid to the cell (pg. 5) can adjust the amount of time that Cas9 is active in the nucleus, thereby selectively limiting off-target editing (pg. 4-6).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the donor nucleic acid sequence rendered obvious by Yoon in view of Wei for a nucleic acid encoding a AcrIIA4 RNA programmable nuclease inhibitor, as described by Shin. A person of ordinary skill in the art would have been motivated to do so in order to selectively limit off-target editing once the donor DNA molecule is integrated into a genomic region of interest. A person of ordinary skill in the art would have had a reasonable expectation of success because both Yoon in view of Wei teach the use of a donor DNA molecule that can be integrated into a genomic region of interest while Shin teaches the use of a nucleic acid encoding a AcrIIA4 inhibitor that can limit Cas9’s genome editing ability in order to selectively limit off-target editing.
Response to Arguments
Applicant's arguments filed 27 October 2025 have been fully considered but they are not persuasive because the newly filed 35 USC 103 rejections of record above were necessitated by amendment. Accordingly, Applicant’s arguments are moot because they are not directed towards the newly pending 35 USC 103 rejections of record above.
Insofar as Applicant’s arguments pertain to the newly recited 35 USC 103 rejections of record, Applicant alleges that Yoon does not teach the use of four separate guide RNAs (Remarks; pg. 8). Applicant alleges that Yoon fails to teach each and every limitation present in the newly amended claims (Remarks; pg. 8).
These argument, and the amendments filed 27 October 2025, are found persuasive. Accordingly, the previously pending 35 USC 102 rejection of record, filed 27 June 2025, have been withdrawn. However, Applicant’s amendments have necessitated the newly filed 35 USC 103 rejections above.
Applicant alleges that Yoon only teaches the use of a single guide RNA to program the claimed DBD (pg. 8).
This argument is not found persuasive because, as described above and as depicted in FIG. 4 of Yoon, Yoon teaches that two CRISPR Cas9 fusion enzymes, each utilizing their own guide RNAs (i.e., first and second gRNAs), can target two genomic DNA sites such that two double stranded breaks (i.e., breaks that create a lesion between first and second genomic sites) are induced in target genomic DNA followed by exonuclease activity in order to insert a donor DNA molecule of interest at the site generated via the two double-stranded breaks ([00299]; see FIG. 4). Thus, Yoon does teach the use of two guide RNA molecules, each directed to different genomic sites and able to guide two different Cas9 fusion proteins to the sites, that can be utilized to create two double-stranded break sites in order to insert a donor DNA of interest.
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached M-R 8:30-4:30, every other F 8:30-4:30 (EDT/EST).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil Hammell can be reached at 571-270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636