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 15 June 2026. Claims 2-6, 8-9, and 11-29 are currently pending. Accordingly, claims 2-6, 8-9, and 11-29 are examined herein.
Any rejection 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 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) 2-6, 8-9, 11-14, 18-22, 23, and 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Bialkowska ("Improved tagging strategy for protein identification in mammalian cells." BMC genomics 6.1 (2005): 113).
Regarding claim 18, Suzuki is directed towards a study concerned with in vivo genome editing via CRISPR/Cas9 mediated homology-independent integration (Abstract). Suzuki teaches the use of homology-independent targeted integration (i.e., HITI) vectors that can be utilized in a method of homology-independent integration of a target gene of interest into a target cell’s chromosome (see Extended Data Figure 1). Suzuki teaches that the HITI vectors function via the use of Cas9 gRNA target sites present on a chromosomal region of interest and Cas9 gRNA target sites flanking a gene of interest present on a donor DNA such that NHEJ repair and homology-independent targeted integration of the gene of interest can occur at the chromosomal region of interest (pg. 144; see Extended Data Figure 1). Suzuki teaches a method of integrating an exogenous gene of interest into a first target intronic region of a chromosome in a first cell comprising delivering an HITI-AAV encoding a Cas9 protein, an AAV-rMertk-HITI donor plasmid comprising an exon 2 of a Mertk gene flanked by two gRNA target sites, and an intron 1 comprising two Cas9 gRNA target sites (pg. 146-148; see Figure 3). Suzuki teaches that vectors encoding gRNAs can be utilized to deliver Cas9 gRNAs to cells of interest (pg. 150; see Extended Data Figure 6). Suzuki teaches that the exon 2 was able to be integrated at intron 1 in order to restore MERTK function in the eye compared to a control vector that utilized homology arms flanking the exon 2 (pg. 148). Suzuki teaches that HITI vectors can be utilized to knock-in Luc or GFP reporter protein into a genomic region of interest to generate knock-in reporters for tracing cells in live animals. (see Extended Data Figure 6).
As evidenced by Liu (Molecular cell 65.2 (2017): 310-322), the Cas9 gRNAs of Suzuki are sgRNAs. Liu is directed towards a review study concerned with how different CRISPR enzymes cleave target nucleic acids of interest (Abstract). Liu teaches that Cas9 associates with a dual-guide RNA structure consisting of a crRNA and a trans-activating CRISPR RNA (tracrRNA) that combine to generate an sgRNA that can direct the Cas9 to a target nucleic acid of interest (pg. 310, 320; see Figure 7).
Suzuki does not teach or suggest that the reporter protein is flanked, 5’ to 3, by a splice acceptor sequence and a splice donor sequence (Claim 18).
Bialkowska is drawn towards a study concerned with an improved tagging strategy for protein identification in mammalian cells (Abstract). Bialkowska teaches the use of an artificial exon (i.e., a donor DNA) comprising, from 5’ to 3, a splice acceptor sequence, a loxP site, a sequence encoding a drug resistance marker (i.e., BSD), a second loxP site, a sequence encoding EGFP (i.e., a reporter), and a splice donor site (i.e., a donor nucleic acid comprising, 5’ to 3’, a splice acceptor site, a sequence encoding a reporter protein, and a splice donor site) (pg. 2; see Figure 1). Bialkowska teaches that insertion of the artificial exon within introns of cellular genes results in expression of hybrid proteins consisting of the tag sequence fused in-frame to sequences of a cellular protein (Abstract). Bialkowska teaches that the BSD sequence lacks a translation initiation codon and harbors two consecutive stop codons which allowed for selection of cells expressing fused proteins by drug selection (pg. 2). Bialkowska teaches that upon providing BSD-positive cells with Cre recombinase, the BSD resistance gene is deleted which leaves behind an EGFP-encoding region, which ultimately leads to the expression of EGFP fused to an endogenous protein (i.e., the integration of the exogenous DNA sequence results in expression of a fusion protein comprising the reporter protein and the protein encoded by the first target gene) (pg. 2; see Figure 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the nucleic acid encoding exon 2 of Suzuki for the artificial exon described in Bialkowska because it would have merely amounted to a simple substitution of one known element for another to obtain predictable results. Because both Suzuki and Bialkowska teach the use of artificial exons that can be inserted within, and expressed from, a target genome comprising introns such that the artificial exons can be expressed within the endogenous genome, one would have expected that utilizing the artificial exon of Bialkowska within the construct of Suzuki to have predictably resulted in the generation of a fusion protein comprising an endogenous protein fused to an EGFP reporter protein. Additionally, because Bialkowska teaches that the resulting insertion of the artificial exon allows for the generation of a functional fusion protein, one would have expected that utilizing the artificial exon of Bialkowska would have resulted in the expression of an EGFP fused to the endogenous protein of Suzuki.
Regarding claim 2, Suzuki teaches that the first and second binding sites may be the same “Scramble” guide RNA binding site (see Methods and Extended Data Figure 1).
Regarding claim 3, Bialkowska teaches that the reporter protein is a fluorescent GFP protein (pg. 5).
Regarding claim 4, Suzuki teaches the gRNA target sites on the donor molecule (i.e., the first and second sgRNA binding sites) are the same gRNA target sites, termed “Scramble” (pg. 150; see Extended Data Figure 1)
Regarding claims 5-6 and 21-22, Bialkowska teaches that the BSD sequence is a blasticidin sequence that is positioned downstream of the splice donor site and the splice acceptor site (i.e., between the splice donor site and the second sgRNA binding site rendered obvious above) (pg. 2). Bialkowska teaches that the BSD sequence is utilized to allow for selection of cells expressing fused proteins by drug selection (i.e., the blasticidin antibiotic resistance gene expression is detected through the drug selection) (pg. 2).
Regarding claims 8-9, Suzuki teaches that the endonuclease is a Cas9 endonuclease (pg. 146-148; see Figure 3).
Regarding claims 11-14, as evidenced by Liu above, the gRNA of Suzuki comprises a fusion of a crRNA and a tracrRNA (pg. 310, 320; see Figure 7).
Regarding claim 19, Suzuki teaches that the exon flanked by gRNA binding sites was able to be integrated at an intronic genomic sequence of a first target gene (pg. 146-148; see Figure 3).
Regarding claim 20, Bialkowska teaches that the reporter protein is expressed when a protein that it was spliced to is expressed (pg. 4; see Figure 2).
Regarding claim 23, Suzuki teaches that reporter proteins can be detected via the use of immunofluorescence images (see Extended Data Figure 1).
Regarding claim 29, Suzuki teaches that the donor plasmid does not comprise a nucleotide sequence having homology arms to the intronic genomic sequence (pg. 144; see Extended Data Figure 1).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Bialkowska ("Improved tagging strategy for protein identification in mammalian cells." BMC genomics 6.1 (2005): 113) as applied to claims 2-6, 8-9, 11-14, 18-22, 23, and 29 above, and further in view of Kamiyama (Nature communications 7.1 (2016): 11046).
Regarding claim 4, Suzuki, as evidenced by Liu, in view of Bialkowska renders obvious claims 2-6, 8-9, 11-14, 18-22, 23, and 29 as described above.
Suzuki, as evidenced by Liu, in view of Bialkowska does not teach or suggest that the reporter is a split fluorescent protein fragment (Claim 4).
Kamiyama is drawn to a study concerned with the use of split fluorescent proteins (Abstract). Kamiyama teaches the use of a nucleic acid encoding a functional split fluorescent mCherry reporter protein (Abstract). Kamiyama teaches that small size of the split fluorescent tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated repair (Abstract).
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 reporter rendered obvious by Suzuki, as evidenced by Liu, in view of Bialkowska for a split fluorescent mCherry reporter protein because it would have merely amounted to a simple substitution of one known element for another to obtain predictable results. Because both Suzuki and Kamiyama teach the integration of sequence encoding reporter proteins into endogenous regions of interest within a target genome, one would have expected that utilizing the nucleic acid encoding the split fluorescent reporter of Kamiyama within the construct of Suzuki, as evidenced by Liu, in view of Bialkowska to have successfully resulted in the insertion and expression of a fusion protein comprising the split reporter and an endogenous protein of interest. Additionally, because Kamiyama teaches that the utilization of the small size of the split fluorescent reporter is advantageous because it is cost effective and easily scalable, one would have been motivated to have done so.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Bialkowska ("Improved tagging strategy for protein identification in mammalian cells." BMC genomics 6.1 (2005): 113) as applied to claims 2-6, 8-9, 11-14, 18-22, 23, and 29 above, and further in view of Liu (Journal of controlled release 266 (2017): 17-26).
Regarding claims 25-28, Suzuki, as evidenced by Liu, in view of Bialkowska renders obvious claims 2-6, 8-9, 11-14, 18-22, 23, and 29 as described above.
Suzuki, as evidenced by Liu, in view of Bialkowska does not teach or suggest that the endonuclease and donor-specific guide RNAs are encoded on a single nucleic acid molecule (Claim 15). Suzuki, as evidenced by Liu, in view of Bialkowska does not teach or suggest that the endonuclease, the guide RNAs, and the donor plasmids are present on a single nucleic acid molecule (Claim 16).
Liu is drawn to a review concerned with CRISPR-Cas9 genome editing systems (Abstract). Liu teaches the use of a plasmid-based CRISPR-Cas9 system that encodes a Cas9 protein and sgRNA on the same vector (pg. 19). Liu teaches that a donor DNA molecule can be utilized alongside the CRISPR-Cas9 system in order to insert the donor sequence in a target genomic sequence of choice (pg. 19; see Fig. 3). Liu teaches that a single plasmid approach is advantageous as it avoids multiple transfections of different components (pg. 19).
Therefore, 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 Suzuki, as evidenced by Liu, in view of Bialkowska such that the endonuclease, donor-specific guide RNAs, and donor DNA are encoded on a single nucleic acid molecule because it would have merely amounted to a combination of prior art elements according to known methods to yield predictable results. Because Liu teaches that utilizing a single plasmid approach allows for the expression and utilization of a CRISPR Cas9 system in an identical way as described in the disclosure of Suzuki, namely the insertion of a donor DNA to a genomic region of interest, one would have expected utilizing a single nucleic acid molecule would have predictably resulted in the insertion of the donor sequence rendered obvious above as applied to claim 18. Additionally, because Liu teaches that a reduction in the number of total transfected plasmids is advantageous, one would have been motivated to have done so.
Claim(s) 17 is/are rejected under 35 U.S.C. 103 as being unpatentable Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Bialkowska ("Improved tagging strategy for protein identification in mammalian cells." BMC genomics 6.1 (2005): 113) as applied to claims 2-6, 8-9, 11-14, 18-22, 23, and 29 above, and further in view of Shin (Biochemical and biophysical research communications 502.1 (7 July 2018): 116-122).
Regarding claim 17, Suzuki, as evidenced by Liu, in view of Bialkowska renders obvious claims 2-6, 8-9, 11-14, 18-22, 23, and 29 as described above.
Suzuki, as evidenced by Liu, in view of Bialkowska does not teach or suggest that the endonuclease-encoding nucleic acid sequence, the donor plasmid, the first site-specific intron targeting guide RNA, and the donor plasmid-specific gRNA encoding sequence are present on separate nucleic acid molecules (Claim 17).
Shin is drawn to a study concerned with targeted sequence substitution through the use of microhomology-mediated end joining (i.e., “MMEJ”) (Abstract). Shin teaches the use of a Cas9 (i.e., an endonuclease) encoding nucleic acid sequence, a vector comprising a donor sequence flanked by two sgRNA binding sites, and two different guide RNAs that target a genomic region of interest such that the donor sequence is integrated into the genome of the target cell (pg. 117; see FIG. 1). Shin teaches that vectors encoding the guide RNAs were co-transfected with a vector encoding a Cas9 endonuclease (pg. 118). Shin teaches that the cells were transfected with mixtures of plasmids encoding a Cas9-red fluorescent protein (RFP)-puromycin expression cassettes, gRNA-expressing plasmids, and donor constructs at a 1:1:10 M ratio using Lipofectamine (i.e., Shin teaches that the endonuclease-encoding sequence, the donor plasmid, and the sequences encoding the sgRNAs are present on separate nucleic acid molecules (pg. 117).
Therefore, 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 vectors rendered obvious by Suzuki, as evidenced by Liu, in view of Bialkowska for vectors wherein the endonuclease-encoding nucleic acid sequence, the donor plasmid, the first site-specific intron targeting guide RNA, and the donor plasmid-specific gRNA encoding sequence are present on separate nucleic acid molecules because it would have merely amounted to a combination of prior art elements according to known methods to yield predictable results. Because Shin teaches that utilizing multiple different plasmids allows for an alternative means of delivery and expression of a CRISPR-Cas9 system in order to insert a donor DNA sequence into a genomic region of interest (i.e., a function shared with the disclosure of Suzuki), one would have expected that utilizing the multi-plasmid approach of Shin within the disclosure of Suzuki to have similarly allowed for the insertion of the donor nucleic acid sequence rendered obvious above as applied to claim 18. Additionally, because Shin provides evidence that the multi-plasmid approach can be successfully utilized to insert and express a reporter protein from a genomic region of interest, one would have expected that the donor nucleic acid sequence rendered obvious above as applied to claim 18 would have also been able to be expressed as a fusion protein when delivered to the genomic region of interest through the use of multiple plasmids.
Claims 24-28 are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Bialkowska ("Improved tagging strategy for protein identification in mammalian cells." BMC genomics 6.1 (2005): 113) as applied to claims 2-6, 8-9, 11-14, 18-22, 23, and 29 above, and further in view of Li (Nature plants 2.10 (2016): 1-6).
Regarding claims 24-28, Suzuki, as evidenced by Liu, in view of Bialkowska renders obvious claims 2-6, 8-9, 11-14, 18-22, 23, and 29 as described above.
Suzuki, as evidenced by Liu, in view of Bialkowska does not teach or suggest integrating the exogenous DNA sequence into an intronic genomic sequence of a second target gene in a second cell by the delivery of the claimed composition and a second site-specific intron-targeting guide RNA that targets a second intronic site in a second target gene (Claim 24). Suzuki, as evidenced by Liu, in view of Bialkowska does not teach or suggest that the exogenous DNA is integrated in two or more cells, wherein the intronic genomic sequence is unique for each of the two or more cells (Claim 25). Suzuki, as evidenced by Liu, in view of Bialkowska does not teach or suggest that the first and second site-specific intron- targeting guide RNA-encoding nucleic acid sequences each target a different intronic site in the first target gene (Claim 26), each target a different intron in the first target gene (Claim 27), or each target a different site in the same intron of the first target gene (Claim 28).
Li is drawn to a study concerned with insertions in different introns in rice via the use of CRISPR- Cas9 (Abstract). Li teaches that guide RNAs can be designed to target different introns in a target EPSPS rice gene (Abstract). Li teaches that the guide RNAs allowed for editing of both the C3 and C5 introns in the target EPSPS rice gene (i.e., Li teaches the use of guide RNAs that are designed such that they different intronic sites and introns in a target gene) (pg. 2; see Figure 1). Li also teaches that multiple base pairs (i.e., different target sites) can be targeted on a singular intron through the use of guide RNAs (pg. 2; see Figure 1). Li teaches that by targeting specific and different intronic sites in different cells, exogenous DNA can be successfully inserted with a high degree of accuracy into target introns (Abstract).
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 at least one set of the genome-specific gRNA molecules described in Suzuki, as evidenced by Liu, in view of Bialkowska for gRNA molecules targeting a different intronic site in first and second target the first target gene, a different intron in the first target gene, and a different site in the same intron of the first target gene because it would have merely amounted to a simple substitution of one known element for another to obtain predictable results. Because both Suzuki and Li teach the use of gRNA molecules that can target intronic regions of interest within a genome of interest in order to insert exogenous DNA, one would have expected that utilizing the gRNA molecules of Li
Response to Arguments
Applicant's arguments filed 15 June 2026 have been fully considered but they are not persuasive.
Insofar as Applicant’s arguments pertain to the previously utilized Korona reference, Applicant’s arguments have been considered but are moot in view of the newly cited Bialkowska reference above which renders obvious the incorporation of a reporter protein into a full-length target gene protein via splicing to produce a fusion protein.
Insofar as Applicant’s arguments are applicable to the newly recited rejections of record, Applicant alleges that the HITI construct of Suzuki is a gene-specific construct and not homology independent because the HITI constructs were designed to insert a GFP cassette downstream of a Tubb3 gene (Remarks; pg. 2).
This augment is not found persuasive because the example to which Applicant is making reference to within Suzuki is drawn towards the use of a HITI vector specifically designed to comprise homology arms in order to compare the construct to a HITI vector that does not comprise homology arms (pg. 144, 150). Suzuki teaches that the study “observed higher knock-in efficiency with HITI donors (IRESmCherry-1c, -2c and –MC; see below for definitions) than with an HDR donor (truncated GFP (tGFP) and IRESmCherry-HDR-0c), a PITCh donor (IRESmCherry-MH) or a HITI donor with homology arms (IRESmCherry-HDR-2c)” (pg. 144). Suzuki also provides working examples of HITI vectors that are homology independent and provides a description of how the donor DNA is inserted into the target gene without homology arms (see Supplementary Figure 1). Thus, the HITI vector that Applicant is making reference to was merely a reference HITI vector that was designed to have homology arms in order to compare it to the HITI vector not comprising homology arms.
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 Mon-Fri, 9AM-5PM (EDT/EST).
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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