DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 08 January 2026 has been entered.
Status of Application
Claims 1-17 remain pending; Claim 14-17 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Claims 1-13 remain subject to examination on the merits.
Terminal Disclaimer
The terminal disclaimer filed on 09 July 2025 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Patent 11242542 has been reviewed and is accepted. The terminal disclaimer has been recorded.
Withdrawal of Previous Objections/Rejections
The objections to the specification for embedded hyperlinks in paragraphs [0012] and [0042] and for use of the Trademark Alt-R ® are withdrawn in view of the amendments the specification to rectify both issues.
The rejection of claims 1-13 under 35 U.S.C. 103 as being unpatentable over Kim et al. (US 2020/0149022 – cited previously) and further in view of Airaksinen et al. (Nuc. Acids. Res., 1998 – cited previously) and Siloto et al. (Biocat. Ag. Biotech., 2012 – cited previously) is withdrawn upon further consideration. Kim et al. provide an enormous list of potential substitutions which may or may not increase on targeting, while only actually testing a fraction of said suggested substitutions. Thus, there would be no specific suggestion or motivation to pick any of the ones not tested with any kind of reasonable expectation of success that the increased targeting activity could be achieved.
Maintained Objections/Rejections
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, 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.
Claims 1-13 are rejected under 35 U.S.C. 103 as being unpatentable over Cereseto et al. (US 2020/0149020 – cited previously) and further in view of Airaksinen et al. (Nuc. Acids. Res., 1998 – cited previously) and Siloto et al. (Biocat. Ag. Biotech., 2012 – cited previously).
Cereseto et al. teach:
Regarding claims 1 and 6, a S. pyogenes Cas9 mutant protein having a single substitution mutation at position L683 and N690 (See claim 1) and D686 – See Table 1, of wild-type SEQ ID NO: 1, wherein said SEQ ID NO 1 has 100% sequence identity with instant SEQ ID NO: 5 (See Supplemental Content, .rapbm file, Duplicates for Result #1), wherein the mutant Cas9 proteins has reduced off-target activity compared to the wild-type SpCas9 (See paragraphs 0010, Figures 8-10).
Regarding claims 2-4 and 10-12, the system utilizes sgRNA (e.g. possesses a crRNA and tracrRNA) – See paragraph 0093.
Regarding claims 7-9, the endonuclease system is encoded by a DNA vector such as a plasmid vector, or a vector suitable for prokaryotic or eukaryotic expression (See paragraph 0058).
Cereseto et al., however, do not teach wherein the mutations at L683 is A, D686 is A or N690 is A (which would be instant SEQ ID NOs: 144, 147 and 150, respectively).
The reference of Airaksinen et al. teaches a method of site-saturation mutagenesis, wherein a single amino acid can be replaced by any other amino acid, which, according to the reference is useful for studying a small, well conserved region of a protein “that is assumed or known to have a functional role” (p. 576, left column). The reference provides advantages of using site-saturation mutagenesis over simple site-directed mutagenesis, namely “several different amino acid substitutions are gained by the same effort” and “additional information can be obtained concerning the nature of acceptable substitutive amino acids” (p. 576, left column). The reference of Siloto et al. is cited as demonstrating the use of site-saturation mutagenesis for directed evolution studies and structure-function studies and analyzing the functional role of a particular amino acid in a protein, wherein various amino acids for differing enzymes were replaced with all possible combinations of amino acids resulting in altered characteristics with respect to acidity, basicity, aromaticity, hydrophobicity, and volume (See Introduction and Sections 3.1-3.3).
Therefore it would have been obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Cereseto et al. with that of Airaksinen et al. and Siloto et al. to perform site-saturation mutagenesis of SpCas9 including substitutions with alanine at the positions taught by Cereseto et al., such as SpCas9 L683, D686 and N690 (among others) in an effort to find mutants with the highest specificity and lowest off-target activity. Altering SpCas9 L683, D686 or N690 (among others) to any of the other amino acids, including alanine, would be well within the skill and knowledge of one skilled in the art as they are simple substitutions. Furthermore, one would have been additionally motivated to mutate these specific amino acids to an alanine or any of the 19 other amino acids and to use site-saturation mutagenesis over simple site-directed mutagenesis to replace the specific amino acids because, as noted by Airaksinen et al. and Siloto et al., site-saturation mutagenesis has advantages over simple site-directed mutagenesis, especially when the region is well known. One would have a reasonable expectation of success to perform site-saturation mutagenesis of these positions SpCas9 is so well known as demonstrated by Cereseto et al. who base their mutations upon the 3-D crystal structure (See paragraph 0092). MPEP 2131.02 states that if the selected combinations of a genus (here any of the other 19 amino acids at a particular amino acid location) can be “at once envisaged”, then the species that fall within that genus are obvious or anticipated. Finally, when the substitutions are made in the wild-type SEQ ID NO: 1 of Cereseto et al., for example at position L683A, D686 or N690, this would result in instant SEQ ID NO: 144, 147 and 150, respectively.
Applicant’s Response and Examiner Rebuttal
Applicant’s traverse the rejection made under 35 U.S.C. 103 as unpatentable over Cereseto in view Airaksinen et al. and Siloto et al.
It is argued that while Cereseto does teach mutations screening based on 3D structural modeling, this does not provide a teaching, suggestion or motivation to generate particular alanine substitutions as recited in the claims, or expect that these substitutions would yield beneficial combination of off-target activity and preserved on-target activity. It is further suggested that Airaksinen et al. and Siloto et al. do not cure this deficiency because they provide no specific guidance or motivation to target L683, D686 and N690. In addition, they both are generic in their disclosures and do not discuss or relate to Cas9 and CRISPR editing systems. In addition, the reliance on Airaksinen et al. and Siloto et al. as secondary references do no suggest specifically changing the positions to alanine. Rather site saturated mutagenesis empirically determines functional outcomes because the outcomes cannot be determined a priori (See Remarks, pp. 9-10). In addition, obvious to try fails as rationale because there are no predictable outcomes and the whole reason for scanning mutagenesis to determine the outcome afterwards in a trial and error fashion (Remarks p. 11).
The Examiner first notes, however, the rejection focuses on teaching, suggestion and motivation as the rationale for the rejection and not obvious to try.
The Examiner has considered these arguments but does not find them convincing. As noted in the pervious Office action response, Cereseto et al. is specifically dedicated to finding alternative SpCas9 variants which are high fidelity variants having increased on/off target ratios (“To address the limitations deriving from the unspecific genomic cleavages of the Streptococcus pyogenes Cas9 (SpCas9) and to identify variants with higher cleavage fidelity, the present invention describes a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets. The screening of SpCas9 variants obtained by random mutagenesis of the Red-II domain allowed the identification of hits with increased on/off ratios. The best performing nuclease, evoCas9, was isolated through the combination of the identified mutations within a single variant. Side by side analyses with previously reported rationally designed variants demonstrated a significant improvement in fidelity of evoCas9 of the present invention.” – Abstract). Thus, through their careful screening of different variants, they identified those with the best on/off target ratios and three of these were identified as L683 and N690 (See claim 1) and D686 – See Table 1. Thus, one skilled in the art is not arbitrarily choosing amino acids of SpCas9 and stumbling upon them, rather Cereseto et al. guides the skilled artisan to these exact identified positions which have been identified as having increased on/off ratios. This, then does provide the motivation to explore specific and various substitutions at all of these positions. The only detail lacking from Cereseto et al. then is which amino acid to change them to and this is remedied by the teachings of Airaksinen et al. and Siloto et al. who specifically teach that site-saturated mutagenesis is highly useful to utilize when specific amino acids or regions of interest have already been identified for substitution(s). The claims require only a single substitution at positions N690A, F682A, L683A, K684A or D686A; and employment of site-saturated mutagenesis for each of these positions will necessarily arrive at singe substitutions including L683A, D686A or N690A, and furthermore, when one does arrive at these specific single amino acid substitutions, this necessarily then gives rise the claimed function of reduced off-target editing activity and maintained on-target editing activity. This is ultimately the same goal as Cereseto et al. (See above and at Abstract “The screening of SpCas9 variants obtained by random mutagenesis of the Red-II domain allowed the identification of hits with increased on/off ratios.”). With regard to Applicant’s assertion that there is no a priori predictability of success, this is not found convincing because the ordinary level of skill in the art (here a graduate student and above) can successfully and easily perform such a site-saturated mutagenesis on a very, very well known enzyme and easily arrive with a substitution of every amino acid including alanine. What the functional outcome of each substitution is not known, but that is the point of the scanning mutagenesis. Required absolute predictability of success and knowing the outcome before doing an experiment is not the legal requirement, See In re Kubin 561 F.3d 1351 (Fed. Circ. 2009) - “[o]bviousness does not require absolute predictability of success . . . all that is required is a reasonable expectation of success.” And MPEP 2143.02. Here, Cereseto et al. already teach positions including L683, D686 or N690 that have been identified to have direct effects on the on-off target ratios, as such a reasonable expectation of success for substituting and identifying the most advantageous ones would be expected, and during the screening, as noted, alanine will definitively be one of the amino acids that is put at these positions.
Finally, regarding Applications assertion of unexpected results as in the specification and Figure 17 (See p 12) is not found convincing because Cereseto have already identified these positions as having increased cleavage fidelity and on/off target activity and then Applicant’s arriving at the same conclusion is not unexpected at all.
For these reasons, the rejection is maintained.
Conclusion
No claim is allowed.
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/SUZANNE M NOAKES/Primary Examiner, Art Unit 1656 23 February 2026