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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 6/23/2026 has been entered.
Application Status
This action is written in response to applicant’s correspondence received on 6/23/2026. Claims 31,40,49-50,64-68,70-72 and 79 are pending. Claim 31 has been amended. Claims 1-30, 32-39, 41-48, 51-63, 69, and 73-78 have been cancelled. Claims 49-50 have been withdrawn from consideration. Claims 31, 40, 64-68, 70-72 and 79 are currently under examination.
Claim Rejections - 35 USC § 101 – New Rejection
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Section 33(a) of the America Invents Act reads as follows:
Notwithstanding any other provision of law, no patent may issue on a claim directed to or encompassing a human organism.
Claim 40 is rejected under 35 U.S.C. 101 and section 33(a) of the America Invents Act as being directed to or encompassing a human organism. See also Animals - Patentability, 1077 Off. Gaz. Pat. Office 24 (April 21, 1987) (indicating that human organisms are excluded from the scope of patentable subject matter under 35 U.S.C. 101).
Specifically, claim 40 recites “a cell comprising the gene editing system of claim 31”. While claims immediately considered on merits recite a composition, the specification describes methods of treating a subject where the claimed cell comprising the gene editing system will be administered for treating and preventing a disease in a human subject (e.g., see page 21, paragraph 6, page 23, paragraphs 2-3, page 170, first paragraph ). Furthermore, claims recite a method of modifying a target nucleic acid in a cell. The specification describes such applications as modifying cells ex vivo and administering the cells to a subject, where a “subject” can be human (page 21, paragraph 6, page 170, first paragraph). Accordingly, when the claimed gene editing system compositions in cells are delivered to a human subject, cells of the subject will comprise the composition claimed. Therefore, the claims would encompass cells in a human organism and the human organism itself.
Amending the claim to an isolated host cell or a host cell in vitro will be remedial.
Claim Rejections - 35 USC § 112 - New Rejection Necessitated by Amendment
Claims 31, 40, 64-68, 70-72, and 79 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Regents of the University of California v. Eli Lilly & Co, 119 F.3d at 1568, 43 USPQ2d at 1406.
Regarding claim 1, claim 1 recites a gene editing system which comprises a template nucleic acid that is between “about” 100-200 base pairs in length which can be either single or double-stranded. The specification defines a “template nucleic acid” as being equivalent to a donor nucleic acid, which can be used in conjunction with CRISPR Cas systems to modify a targeted gene (page 14, third paragraph). Thus, the template nucleic acids are recited with structural-functional relationships in that they are part of a “gene editing system” where “template nucleic acids” are definitionally required to be able to target or modify a target gene. Claim 1 is broadly encompassing the genus of template nucleic acid which are “about” 100 nucleotides in length, which reasonably includes embodiments of template nucleic acids which are 80 nucleotides in length as no definition of “about” is offered in the specification. Claim 1 is problematic because the Applicant has not demonstrated that such template nucleic acids that are single-stranded and as short as “about” 100 nucleotides are functional in gene editing or as “template nucleic acids” which as presently defined in the specification include the function of being able to target DNA as donor templates (page 14, third paragraph). The Applicant was therefore not in possession of the claimed scope of the genus “template nucleic acid” because the genus is known in the art to be unpredictable and non-functional at shorter lengths encompassed by the claims (see below).
Regarding the guidance provided in the specification, the Applicant has tested template nucleic acids which are 129 and 179 nucleotides in length, and shown that shorter nucleotides perform significantly worse as templates for gene correction (Figures 3 and 4). The Applicant has not tested or demonstrated that templates which are “about” 100 nucleotides, for instance, 75 nucleotides, can in fact function with gene editing capacities, where shorter single-stranded templates were not tested. This is problematic because it is known in the art that shorter single-stranded templates in similar systems as those recited do not function to target DNA (see below).
Regarding the state of the art, it is known in the art that template nucleic acids which are single-stranded are known to be non-functional in gRNA-template fusion systems such as those presently recited. For instance, Potter (WO 2016/065364 A1, provided in an IDS) is a patent document which teaches gRNA-template fusion systems similar to those presently recited (Title, Abstract, throughout, Figures 1, 7, and 9). Potter teaches that they tested a single-stranded 80 basepair donor template molecule, where furthermore it was shown that the 80 bp ssDNA template did not function to target/edit a target DNA (page 26, first paragraph, paragraph 129). Per Figure 11, the ssDNA conjugate did not function to introduce a restriction cleavage site a target site (see Figure 11 of Potter and paragraph 129). Thus, Potter has tested template DNA which are “about 100” nucleotides and shown them to be non-functional (page 26, first paragraph 129, Figure 11).
The Applicant was not in possession of the genus of template nucleic acid claimed because they have not demonstrated that short, single-stranded nucleic acids can effectively function in “gene editing” systems as claimed, or as “template nucleic acids” which are defined to be able to target DNA. Further, Potter teaches that such template nucleic acids are non-functional when targeting DNA. The Applicant has not demonstrated possession of the lower end of the claimed length of the recited nucleic acid templates (i.e., “about 100”).
Claims 40, 64-68, 70-72, and 79 depend from claim 1 and do not resolve this issue and are therefore also rejected under 112(a).
Claim Rejections - 35 USC § 103 – New Rejection Necessitated by Amendment
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.
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 31, 40, 64-68, 70-72, and 79 are rejected under 35 U.S.C. 103 as being unpatentable over May et al. (WO 2014/150624 A1, of record) in view of Yin (WO 2015/191693 A2, published 12/17/2015, earliest effective filing date 06/10/2014, of record), Potter et al. (WO 2016/065364 A1, earliest effective filing date October 24, 2014, provided in an IDS), Maeder (WO 2015/153791 A1, of record), and Gratz (Gratz SJ et al. Genetics. 2013 Aug;194(4):1029-35). Claim 31 is further evidenced by Dubitzky (Dubitzky et al. Encyclopedia of Systems Biology, DOI 10.1007/978-1-4419-9863-7, published 2013, of record). The rejection is further evidenced by GenBank L29345 (NCBI GenBank Database, accession number L29345.1, GFP genetic sequence, published 12/30/1994, accessed 12/13/2024).
Regarding claim 31, May describe a gene editing system, comprising a gRNA fusion molecule and at least one Cas9 molecule (complex comprising a site-directed polypeptide, a nucleic acid-targeting nucleic acid, and a donor polynucleotide can be delivered to a target nucleic acid; paragraph [0851], nuclease, e.g., Cas9; paragraph [0850]). May et al. describe a gRNA complex molecule, comprising a gRNA molecule and a template nucleic acid (complex comprising a gRNA and a donor polynucleotide; paragraph [0851], figure 30). Furthermore, the final sentence of paragraph 284 of May teaches that the 3’ tracrRNA sequence can comprise one or more hairpins (i.e., at least two hairpins). The 3’ tracrRNA sequence is identified as 130 in Figure 1A (paragraph 284), which clearly depicts this sequence/structure at the 3’ end of the molecule. Furthermore, paragraph 333 teaches that the 3’ tracrRNA sequence comprises a stem loop structure. Thus, May teaches a stem loop structure at the 3’ end of their gRNA molecules. May therefore teaches that the 3’ end of their gRNA molecule comprises one or more hairpin loops (paragraph [0284]). May further teaches that their template nucleic acids comprise single-stranded DNA, or double-stranded DNA (in some embodiments, the donor polynucleotide can be single stranded. In some embodiments, the donor polynucleotide can be double stranded; paragraph [0860], furthermore, May defines a “donor polynucleotide” as a nucleic acid [00159], and states that a nucleic acid can be DNA [00174]; thus, May teaches that their donor/template polynucleotides can be single- or double-stranded DNA).
May also teaches Figure 30, wherein the description of Figure 30 on page 49 states that Figure 30 “depicts exemplary methods of the disclosure of bringing a donor polynucleotide to a modification site in a target nucleic acid”, paragraph 121. Furthermore, paragraph 849-860 of May teaches that their method includes bringing the donor polynucleotide into close proximity of the site directed target by associating, in various ways (Figure 30A-30F) the donor with the gRNA, using hybridization strategies, and thereby enhancing the insertion of the donor polynucleotide into the site by increasing its proximity to the target site by directly linking/attaching it with the gRNA (Figure 30A-30F and paragraphs 849-860). Thus, May teaches enhanced delivery of donor nucleic acids to target sites by directly linking the donor nucleic acid with gRNA.
May teaches that the donor polynucleotide/template is hybridized/annealed to the 3’ end of the gRNA through complimentary base pairing/annealing (Figure 30A-30E) or through RNA/DNA binding protein interactions (Figure 30F). May therefore teaches non-covalent linkage of the gRNA to the template/donor nucleic acid (Figure 30). May teaches that the 5’ end of donor can bind the 3’ end of the gRNA (Figure 30B).
Furthermore, May teaches that site-directed mutations may be made to regions and domains of the Cas9 such as the HNH domain, where such modifications can alter the nuclease activity of the domain (e.g., paragraphs 499-500). May also teaches that mutations may be made in Cas proteins to form nickases, where the nuclease activity of a domain is targeted to form a nickase (e.g., paragraph 602). Furthermore, May teaches that such adoption of nickase strategies can be used for targeting genomic loci, where the target cleavage sites can be within the general region of a target site (e.g., paragraph 604). Furthermore, May teaches that target DNA targeted using nickases can be used, where the gRNA targets 5’ of a target sequence (Figure 34). Per the instant specification, the drawing presently recited in claim 31 broadly includes a nickase Cas9 which nicks between 1-10,000 bases 5’ of a target site (bottom of page 136 into top of page 137 of the present specification). Thus, the teaching of May, who teaches that nickases can cut at a target site or 5’ of a target site for insertions or deletions reasonably reads on the presently recited drawing of target position and cleavage event recited in the drawing of claim 31 (paragraphs 602-607).
May teaches that Cas9 can comprise two or more nuclease domains including RuvC and an HNH domain (paragraph 246). May further teaches that the HNH domain can be modified to contain mutations (section entitled “Modifications to the HNH domain”, paragraphs 547-557). May further teaches that “The nuclease used in the methods of the disclosure (e.g., Cas9) can comprise nickase activity in which the nuclease can introduce single-stranded breaks in a target nucleic acid” paragraph 859. Therefore, May teaches that the Cas9 they used in the study can comprise a RuvC and HNH domain, and also teaches mutations in the HNH domain, and further teaches that the Cas9 they teach can be in the form of a nickase.
May teaches that donor polynucleotides can comprise a reporter element such as GFP (paragraph 769). As evidenced by GenBank L29345, the nucleic acid sequence of GFP is 922 basepairs (page 2). Thus, by teaching that donor polynucleotides can comprise a nucleic acid sequence encoding GFP, which is 922 bp, May teaches that template nucleic acids can 922 bp in length.
May does not teach that the donor polynucleotide is covalently linked with the gRNA in their molecule complexes, or that this covalent linkage is a phosphodiester bond between the 3’ end of the gRNA molecule and the 5’ end of the template nucleic acid. May, while teaching Cas9 nickases which comprise a RuvC domain and an HNH domain that can be inactivated to form the nickase, and teaches a motivation to make such a Cas9 to target DNA, does not specifically reduce the Cas9 nickases to practice which has an inactive HNH domain. May does not teach that the donor template is between 100-200 nucleotides in length.
Yin is a patent document focused on enhancing gene editing tools (paragraph 3). Specifically, Yin teaches methods related to modifying gene sequences using CRISPR/Cas9 editing systems, and delivery systems for achieving gene modification using gRNAs and donor nucleic acids (Abstract, and e.g., paragraphs 6 and 83). The teachings of Yin and May therefore directly overlap in subject matter and stated goals because both relate to the use of gRNA/CRISPR/donor nucleic acid template delivery (see Abstracts of both, and see documents) Yin teaches that the gRNA and repair templates used in their methodology can be covalently linked to each other and non-covalently linked to each other (paragraphs 6, 15, 23, 83, and claims 18, 22, 27, 54 114, and 116). Paragraph 15 of Yin teaches that, while the gRNA and the repair template/donor polynucleotide can be covalently linked, the repair template can also be partially annealed to the gRNA, as was also taught by May (May, Figure 30). Thus, Yin teaches both partial annealing (non-covalent) and covalently linking gRNA with a repair template, and also that these two methods of associating a gRNA and a template/donor/repair template can be used interchangeably as they accomplish the same task, as evidenced by the fact that they are recited in the alternative as methods of linking gRNA with template a nucleic acid (e.g., paragraphs 6 and 83). Additionally, Yin teaches in paragraph 15 that the repair template that is covalently linked with the gRNA can be DNA.
Furthermore, Yin teaches that as a result of linking the gRNA to the repair template DNA, which can be bound either covalently or non-covalently as functional alternatives, the efficiency of nucleotide sequence modification by which the repair template is efficiently directed to the nucleus of the cell is “greatly improved,” (paragraph 83). Thus, Yin teaches that there are two ways to bind gRNA to donor templates: either non-covalently or covalently, that such binding can be used interchangeably, and that a result of linking gRNAs to donor templates is significant improvement of sequence modification (paragraph 83).
Furthermore, Yin teaches that the repair template (i.e., “the template nucleic acid”) can be 200 bp in length, and therefore teaches that the repair template can be “about” 200 nucleotides in length (paragraph 15).
Furthermore, Potter et al. describe a gRNA fusion molecule, comprising a gRNA molecule and a template nucleic acid (donor nucleic acid is covalently linked to guide RNA; paragraph [0036]), wherein the template nucleic acid comprises single-stranded DNA, or double-stranded DNA (donor nucleic acid will typically be DNA and may be single-stranded or double-stranded; paragraph [0050]).
Furthermore, Potter and May directly overlap in subject matter because Potter also teaches gRNAs with 3’ hairpin structures that are attached to donor/template nucleic acids (e.g., Figures 1,7, and 9). Potter also teaches the goal of improving the efficiency of homologous recombination in CRISPR systems for gene editing by increasing the concentration and proximity of donor/template nucleic acids to target sites (Abstract, and paragraphs 2-4). Thus, Potter, May, and Yin are all in the same field of endeavor, use similar reagents and methods, and overlap in objective, aim, and subject matter.
Potter teaches Figures 1, 7, and 9, along with a description of Figure 1 in paragraph [0006] stating that “the hairpin nucleic acid molecule is guide RNA.” Figures 1, 7, and 9 of Potter depict nucleic acid molecules with two regions of secondary structure, wherein one region of secondary structure is in the middle of the molecule and one is at the 3’ end. The structures at the 3’ end of the gRNAs in Figures 1, 7, and 9, contain a region which has folded back (i.e., complimented) with itself, forming a stem structure and, because these regions at the 3’ end contain stem structures, these regions must also inherently contain loop structures, as evidenced by Dubitzky. Dubitzky is an encyclopedia of terms and definitions used in systems biology. Dubitzky teaches that “hairpin structure is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a stem-loop structure”, “Definition”, first paragraph. Dubitzky therefore teaches that the term “hairpin” (which is the term Potter uses to describe the structure in Figure 1 in paragraph 6) is synonymous with stem-loop structure, and corroborates that the hairpin structure recited by Potter inherently contains a stem loop. Furthermore, Dubitzky teaches that “the formation of a hairpin structure is dependent on the stability of the resulting helix and loop regions. The first prerequisite is the presence of a sequence that can fold back on itself to form a paired double helix”, Formation and Stability, first paragraph, and that “the stability of the loop also influences the formation of the hairpin structure. “Loops” that are less than three bases long are sterically impossible and do not form”, “Formation and Stability”, second paragraph. Figures 1, 7, and 9 of Potter clearly depict a gRNA nucleic acid molecule that has a secondary structure at the 3’ end, wherein said secondary structure contains a region that has “folded back” on itself, i.e., has formed a stem structure. Furthermore, as taught by Dubitzky, it is physically impossible for such a “folding back” stem structure to occur with a loop less than three nucleotides in length. Given that the 3’ end of the gRNA molecules depicted by Potter in Figures 1, 7, and 9 clearly contain a region that has complimented with itself to form a stem, and that Dubitzky teaches that it is impossible to have a stem without at least a four base loop, the secondary structure at the 3’ end of Figures 1, 7, and 9 are inherently hairpin loop structures.
Thus, the teachings of May and Potter directly overlap in scope and subject matter because each teach gRNAs linked with template nucleic acids, where the 3’ end of the gRNA molecule comprises a hairpin loop, and where the template nucleic acid comprises single-stranded DNA or double-stranded DNA.
Furthermore, Potter teaches that the 3’ end of their gRNA molecule is linked to the 5’ end of the template nucleic acid by a phosphodiester bond (gRNA-azido-dATP, figures 7 and 9).
Potter teaches that their invention “involves enhancing homologous recombination by increasing the concentration of donor nucleic acid at or in close proximity to the junction of a break in a nucleic acid molecule resident in a cell,” (paragraph 4). Potter therefore teaches that their methods, which use gRNAs comprising 3’ hairpin loops covalently bound to template/donor DNA enhances recombination/gene editing events (paragraph 4). Thus, Potter directly teaches a motive to incorporate their teachings into similar systems, such as those taught by May and Yin (paragraph 4).
Furthermore, Potter, like May, also teaches that either the HNH or RuvC domain of Cas9 can be targeted to inactivate one of these nuclease sites for the beneficial outcome of making a nickase, and that this is a known, useful strategy to generate a nickase version of the protein (paragraph 6). Furthermore, Potter also teaches that, when using such nicakses comprising for instance an inactive HNH domain, a target locus can be cut within the surrounding region of the target (paragraph 31). Thus, Potter also teaches that cleavage sites can be in the surrounding region of a target site, and therefore teaches the targeting strategy depicted in the drawing in claim 31 (paragraph 31, where the specification at pages 136-137 recite that the gRNA can cut within 1-10,000 bps of the target, where Potter’s teaching of cutting in the region of a target reasonably reads on 1-10,000 bases of the target locus, paragraph 31 or Potter).
Furthermore, Potter teaches that the donor DNA can be 480 bp in length, where such a donor was reduced to practice (paragraph 115, Figure 11).
Furthermore, Potter teaches that covalently linking gRNAs with donor templates dramatically increase the efficiency of targeting efficiency:
“To our surprise, the recombination efficiency significantly increased with the
increase of donor DNA with 34% in Jurkat T cells according to sequencing analysis. When a donor DNA was conjugated to a gRNA, the recombination efficiency increased to 75% in Jurkat T cells,” (paragraph 129).
Regarding a method of generating a Cas9 with functional RuvC domain and inactive HNH domain, such a strategy is already known and reduced to practice as taught by Maeder. For instance, Maeder is a patent document related to methods of CRISPR gene editing and targeting (Description). Maeder teaches that “the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at N863, e.g., the N863A mutation”, (page 806, lines 17-19). Therefore, Maeder teaches that Cas9 has a RuvC domain and an HNH domain, and further teaches that a mutation at N863 renders the Cas9 molecule a nickase enzyme with RuvC activity remaining intact while inactivating the HNH domain. Thus, Maeder teaches a known method of rendering a Cas9 protein into a nickase by making a mutation at position N863, which leaves the RuvC domain intact but inactivates the HNH domain. Thus, Maeder teaches that mutational strategies to render Cas9 nickases, where the mutation leaves an active RuvC domain and inactive HNH domain (as taught by both May and Potter) are already known and reduced to practice. Thus, the teachings of May regarding inactivating the HNH domain are predictable and known in the art.
In addition, regarding the limitation that the donor is between 100-200 nucleotides in length, Gratz is a research article that focuses on genome engineering using CRISPR Cas9 systems (Title, Abstract, and throughout). Gratz therefore directly overlaps in subject matter and field of endeavor with May, Yin, Potter, and Maeder because all of these prior art references are concerned with genomic targeting and engineering using the same CRISPR Cas systems (throughout). Gratz teaches that it is known in the art that the donor nucleic acid can be 170 nucleotides in length:
“we designed a single-strand oligodeoxynucleotide (ssODN) repair template that includes a 50-nt attP site flanked by 60-nt homology arms corresponding to sequences 59 and 39 of yellow,” (page 1030, right column final paragraph to page 1031, left column first paragraph and Figure 1)
Furthermore, aside from the fact that Gratz has reduced to practice donor templates which are 170 nucleotides in length (50 nt + 60 (first homology arm) + 60 (second homology arm) = 170 nts (page 1030, right column final paragraph to page 1031, left column first paragraph and Figure 1), Gratz also teaches that such constructs are useful for introducing sites such as attP sites into the genome, where a practitioner can use such sites for a given desired design purpose (page 1030, right column, final paragraph). Thus, a practitioner could immediately envision donor templates which are between 100-200 nucleotides in length, where such donor templates have already been taught and reduced to practice by Gratz, where furthermore such donor templates are customizable for a given application per Gratz (page 1030, right column, final paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the gRNA/donor templates of May with Yin and Potter, to arrive at a gRNA fusion molecule comprising a gRNA covalently linked to a template nucleic acid, wherein the 3’ end of the gRNA molecule comprises two hairpin loops, wherein the 3’ end of the gRNA molecule is linked to the 5’ end of the template nucleic acid by a phosphodiester bond, and wherein the template nucleic acid comprises DNA. The combination of May, Yin, and Potter is the simple substitution of one method of linking gRNA and template DNA (non-covalent annealing, May) for another known method of linking gRNA and template DNA (covalent phosphodiester 3’/5’ linkage, as taught by Potter/Yin) to obtain predictable results. In addition, the combination is also obvious to try, where a practitioner would be choosing from only two options (covalent or non-covalent bonds), where the solution is predictable because both are taught to be viable ways of binding nucleic acid molecules, and further where the expectation of success is reasonable because phosphodiester/covalent bonds are known to work to link nucleic acids (Potter). Furthermore, a practitioner would be motivated to combine the teachings of May, Yin, and Potter because Yin and Potter teach that their methods can greatly improve the efficiency of gene editing (Yin, paragraph 83, Potter paragraphs 4 and 129).
Furthermore, May already teaches atCas9 comprising a RuvC and inactive HNH domain are known, and are useful for targeting DNA regions as a nickase. The product recited in claim 31 is further obvious in view of Maeder, who teaches specific strategies to make the HNH-inactive Cas protein already taught by both May and Potter. Thus, an inactive HNH domain is not a novel feature, nor is it unpredictable as it is well-known in the art as taught by May, Potter, and Maeder, where further a practitioner would be motivated to make such an HNH-inactivation based on the teachings of May so that they could target specific DNA targets with nicakse Cas9 enzymes using the strategy of Maeder.
Furthermore, regarding the drawing in claim 31 depicting a targeting/cleavage strategy, Potter teaches that nickases cleave in the general surrounding area of a target as a known strategy of DNA targeting (paragraph 31). Thus, the targeting/cleavage strategy using an HNH-inactive nickase is simply the known targeting strategy when using such enzymes, as taught by Potter (paragraph31). It would therefore be obvious to a person of ordinary skill in the art to adopt the nickases taught by May to use the targeting strategy as taught by Potter, to arrive at the targeting/cleavage strategy depicted in claim 31.
Additionally, the range of donor template length (100-200) is obvious in view of the teachings of Yin, May, Potter, and Gratz, because Yin already teaches 200 bp donor template length, where furthermore it was known in the art that donor template lengths which are 170 nts in lengths are useful in CRISPR Cas applications and have been reduced to practice as taught by Gratz (above). The results are therefore predictable, where the combination is the simple combination of known prior art elements to yield predictable results. Additionally, such a length of template nucleic acid as that recited in claim 31 (100-200 nucleotides) could be arrived at by routine laboratory optimization, where donor templates within this ranges are already known as taught by Yin and Gratz.
Regarding claim 40, May teaches a cell comprising the gRNA fusion molecule or gene editing system (tagged cell can be generated by contacting the cell with a donor polynucleotide, and a complex comprising a site-directed polypeptide and a nucleic acid-targeting nucleic acid; paragraph [0826]).
Regarding claim 64, May teaches that the 3' tracrRNA sequence can comprise one or more hairpins; paragraph [0284].
Regarding claim 65, May teaches that their nucleic acids can comprise an MS2 binding site (paragraph 741).
Regarding claim 66, May teaches ligation of nucleotides using T4 DNA ligase, paragraph 866. Claim 66 is therefore obvious as it would be obvious to a practitioner of ordinary skill in the art that such nucleic acids could be ligated together, particularly in light of the fact that the gene editing system, comprising covalent linkage and phosphodiester bond is rendered obvious in view of May, Yin, and Potter. Ligation is therefore a known method taught by May of joining nucleic acids together.
Furthermore, claim 66 is not drawn to a method of ligation, but to a product. Thus, recitation of the limitation that the gRNA is ligated to the template nucleic acid does not change the structure of the product recited, as the claim depends from claim 31 and requires covalent linkage with a phosphodiester bond. Thus, even though it would be obvious to use a ligase to join the nucleic acids, the limitations of claim 66 regarding the how the gRNA and template are joined do not add structural limitations to the claim limitations recited in claim 31.
Regarding claims 67, 68, and 71, May teaches that the site-directed polypeptides used in their methods can be enzymatically active (paragraph 611), and further states that “in some embodiments, the polypeptide is Cas9”, paragraph 17.
Regarding claim 70, Maeder teaches that “the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at N863, e.g., the N863A mutation”, (page 806, lines 17-19). May teaches SpCas9 (paragraph 888).
Regarding Claim 72, both May and Potter teach the formation of ribonucleoprotein complexes for Cas/gRNA systems (May paragraph 214, Potter paragraph 101).
Regarding claim 79, Potter also teaches that, when using nicakses comprising for instance an inactive HNH domain, a target locus can be cut within the surrounding region of the target (paragraph 31). Thus, Potter teaches that cleavage sites can be in the surrounding region of a target site, and therefore teaches the targeting strategy depicted in the drawing in claim 31, where Potter’s teaching of cutting in the general region of a target reasonably reads on 1-10,000 bases of the target locus, paragraph 31 or Potter). Potter teaches that when using dual nickase targeting systems, the nicks are made in the surrounding region of the target (i.e., within 1-10,000 basepairs)
It would have been obvious to a person of ordinary skill in the art before the effective filing date to target to design the Cas9 to cleave a single strand (i.e., nick) within 1-10,000 base pairs of the target site because Potter teaches that, when using nickases, this is the known strategy of targeting DNA. Thus, a practitioner would be motivated to nick a site within 1-10,000 basepairs of the target, so that they could carry out the known strategy taught by Potter.
Response to Arguments
The Applicant’s arguments filed 6/23/2026 have been considered but are not persuasive.
The Applicant argues that the prior art fails to teach each of the limitations, and also fails to identify a reason to combine the May, Yin, and Potter references with a reasonable expectation of success. This argument is not found to be persuasive. As will be discussed further below, the prior art references cited do teach all of the recited limitations and furthermore provide sufficient motivation to combine the references. The general thrust of the Applicant’s arguments appear to be piecemeal analysis, where each individual reference is attacked individually for what they teach and/or do not teach. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to applicant's argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007).
In this case, as discussed above, May teaches each of the claim elements of claim 31, with the exception that they do not teach covalently linking the gRNAs with the donor template, the donor template length of 100-200 nucleotides, and does not reduce the Cas9 nickase to practice (although they do teach HNH inactivation where the RuvC domain remains active, see above). However, Yin teaches that linking gRNAs to donor templates by partial annealing/non-covalent linkages is interchangeable with using covalent bonds (Yin, paragraph 83) to accomplish the same task as May (i.e., to bring donor template DNA in proximity to its target). Furthermore, Yin teaches that linking gRNA and donor molecules greatly improves CRISPR/Cas gRNA systems because the repair/donor template can be efficiently directed to its intended location (paragraph 83). The Applicant has therefore not originated the concept of linking gRNAs with donor templates because Yin already teaches this design and its advantages. Yin therefore teaches a strong motivation to link gRNAs to template/donor nucleic acids using either covalent bonds and/or non-covalent bonds as ready, interchangeable alternatives. The Applicant has therefore not invented the concept of covalently linking gRNAs with template donors.
Furthermore, Yin teaches donor templates which are 200 bps in length, and therefore teaches that such template lengths are known in the art.
Potter also teaches the same principle, where increasing the proximity of donor/template DNA by binding it directly to gRNA improves editing efficiency (Summary, Figures 1, 7, and 9). Thus, a practitioner is taught a known motivation to bind gRNA to donor/template DNA, because this is a known strategy to bring the donor template in close proximity to its target (Yin, Potter, May). Potter further teaches a strong motivation to apply their method of covalent linkage because they teach that it dramataically increases recombination efficiency (paragraph 129). Furthermore, Yin teaches that non-covalent and covalent linkages are readily interchangeable while Potter teaches that binding gRNA to donor templates using phosphodiester covalent bonds is a known method (e.g., Figures 1, 7, and 9). In addition, not only does Potter teach covalent binding of gRNA to donor templates using a covalent phosphodiester bond, the gRNA of Potter also contains a 3’ hairpin structure similar to May (Figures 1,7,9 Potter, Figure 30 of May). Thus, there is a reasonable expectation of success in combining May, Yin, and Potter, because these systems are all gRNA donor/template DNA systems which use the same class of molecules to accomplish the same task, where the gRNA of Potter is structurally very similar to that of May (May, Figure 30, Potter, Figures 1, 7, and 9). Thus, contrary to the Applicant’s assertions, a strong motivation and strong level of predictability exists when combining the cited references.
Furthermore, the KSR rationale of a simple substitution of one known art element for another to obtain predictable results is applied with regards to the use of either a non-covalent or covalent bonds (see the rejection of claim 31, above). A conclusion of obviousness based on this rationale is sufficient to show obviousness per MPEP 2141. In the present case, it was already known that covalent bonds can be used to attach gRNAs with donor templates, as taught by Yin. Thus, the KSR rationale is satisfied, as the two bonds (non-covalent and covalent) are functional alternatives of one another in the context of the present invention, a taught by Yin (see 103 rejection, above). Thus, the rejection can be viewed as the simple the substitution of one known element for another with predictable results.
The Applicant argues that Potter teaches an extremely large genus of potential lengths of template donors. This argument is not persuasive, as Yin teaches embodiments which fall within the recited range (200 bp, as the Applicant concedes). Furthermore, Gratz also teaches that useful donor templates as short as 170 bp can be used (see above). The art is therefore replete with knowledge surrounding the template donor’s length, where templates within the range recited (100-200) are already known and reduced to practice in order to achieve DNA targeting, per Gratz.
The Applicant argues that their results are unexpected because the 129 nt template worked with less efficiency than the 179 nt template. This argument is not persuasive. As an initial matter, Potter has already taught that shorter templates are less effective than longer templates (Figure 11, paragraph 129). Thus, the result that the 129 nt template was less effective is not surprising. Furthermore, Gratz teaches templates which are 170 nucleotides which are known to function to target DNA for recombination. The Applicant’s reduction to practice of a 179 donor is therefore not unexpected or surprising because a donor template that is even shorter than this (170, per Gratz) has already been reduced to practice. Thus, the art has already established that there is a lower limit to the functionality of donor templates (Potter, 80 bp), where the art furthermore has already taught functional embodiments of donor nucleic acids within the recited range (170 bp, per Gratz). The result that shorter templates are less functional than longer templates ~170 bp is therefore not surprising, as such teachings were known in the art.
The art was therefore replete with knowledge and motivation to arrive at the presently recited invention.
Conclusion
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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