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 .
Applicant's listing of the claims and arguments filed on 07-16-2026 have been received and entered. Claims 82, 87, 88, 89, 100-101 have been amended. Claims 1-81 and 95-99 have been canceled. Claims 82-94, 100-101 are pending in the instant application.
Election/Restrictions
Applicant's election with traverse of Group I, claims 82-94, 100-101 in the reply filed on 01-05-2026 acknowledged. The response to traversal can be found in the office action mailed on 04-16-2026.
Claims 95-99 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 01-05-2026. It is noted that claims 95-99 have been canceled.
Claims 82-94, 100-101 are under consideration.
Priority
This application is 371 of PCT/EP2021/084213 filed on 12/03/2021 which claim priority from EP 20211801.4 filed on 12/04/2020. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Claim Objections
Claim 101 is objected to because of the following informalities:
It appears there is no item “vii” between items “vi” and “viii”
Appropriate correction is required.
Claim Interpretation
35 U.S.C. 112(f)
The following is a quotation of 35 U.S.C. 112(f):
(f) Element in Claim for a Combination. – An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The following is a quotation of pre-AIA 35 U.S.C. 112, sixth paragraph:
An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
Claims 82-94, 100-101 are being interpreted under 35 U.S.C. 112(f).
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art. The broadest reasonable interpretation of a claim element (also commonly referred to as a claim limitation) is limited by the description in the specification when 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is invoked.
As explained in MPEP § 2181, subsection I, claim limitations that meet the following three-prong test will be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph:
(A) the claim limitation uses the term “means” or “step” or a term used as a substitute for “means” that is a generic placeholder (also called a nonce term or a non-structural term having no specific structural meaning) for performing the claimed function;
(B) the term “means” or “step” or the generic placeholder is modified by functional language, typically, but not always linked by the transition word “for” (e.g., “means for”) or another linking word or phrase, such as “configured to” or “so that”; and
(C) the term “means” or “step” or the generic placeholder is not modified by sufficient structure, material, or acts for performing the claimed function.
Use of the word “means” (or “step”) in a claim with functional language creates a rebuttable presumption that the claim limitation is to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites sufficient structure, material, or acts to entirely perform the recited function.
Absence of the word “means” (or “step”) in a claim creates a rebuttable presumption that the claim limitation is not to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is not interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites function without reciting sufficient structure, material or acts to entirely perform the recited function.
Claim limitations in this application that use the word “means” (or “step”) are being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action. Conversely, claim limitations in this application that do not use the word “means” (or “step”) are not being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action.
In the instant case, claims 82, 89-90 are being interpreted under 35 U.S.C. 112(f) for the following reasons:
Regarding (A), the claim limitations use a term “targeting means” that is a generic placeholder for performing the claimed function.
Regarding (B), the generic placeholder is modified by functional language : “targeting means directing the nuclease to the target nucleic acid sequence” in claim 82; “targeting means from binding to and/or generating a further SSB or a further DSB in the resulting nucleic acid sequence” in claim 89; “targeting means comprise or consist of a guide RNA capable of hybridizing to the genomic target region” in claim 90.
Regarding (C), the generic placeholder is not modified by sufficient structure, material, or acts for performing the claimed function.
Therefore, regarding claims 82, 89-90, the phrases “targeting means directing the nuclease to the target nucleic acid sequence” in claim 82 and “targeting means from binding to and/or generating a further SSB or a further DSB in the resulting nucleic acid sequence” in claim 89 and “targeting means comprise or consist of a guide RNA capable of hybridizing to the genomic target region” in claim 90 are interpreted as a means-function limitation under 35. U.S.C. 112f because it contains the generic placeholders “targeting means” that is modified by the functional language and are not modified by sufficient structure, material, or acts for performing the claimed function because there are no other elements recited in the claim.
It is noted that the specification of the claimed invention teaches that “Targeting means: the term refers to a moiety or a molecule, which enables a nuclease to recognize its target nucleic acid. For a CRISPR/Cas nuclease, the targeting means refers to the guide RNA or crRNA. For TALENs, the targeting means refers to the TAL effector DNA-binding domains. For ZFNs, the targeting means refers to the zinc finger DNA-binding domains. The targeting means may thus be a moiety of the nuclease (for ZFNs and TALENs), or it may be a different molecule altogether (for CRISPR/Cas systems) (see page 21 lines 29-35). Therefore, instant claims 82, 89-90 and their dependent claims 83-88, 91-94, 100-101 are interpreted accordingly.
Maintained in modified form-Claim Rejections - 35 USC § 112 - necessitated by amendments
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 86 and 88 remain rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 86 and 88 recite the term “substantially” which is a term of degree which renders the claims vague and indefinite because it is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. As per MPEP 2173.05(b), when a term of degree is used in the claim, the examiner should determine whether the specification provides some standard for measuring that degree. Hearing Components, Inc. v. Shure Inc., 600 F.3d 1357, 1367, 94 USPQ2d 1385, 1391 (Fed. Cir. 2010); Enzo Biochem, Inc., v. Applera Corp., 599 F.3d 1325, 1332, 94 USPQ2d 1321, 1326 (Fed. Cir. 2010); Seattle Box Co., Inc. v. Indus. Crating & Packing, Inc., 731 F.2d 818, 826, 221 USPQ 568, 574 (Fed. Cir. 1984). If the specification does not provide some standard for measuring that degree, a determination must be made as to whether one of ordinary skill in the art could nevertheless ascertain the scope of the claim (e.g., a standard that is recognized in the art for measuring the meaning of the term of degree). For example, in Ex parte Oetiker, 23 USPQ2d 1641 (Bd. Pat. App. & Inter. 1992), the phrases "relatively shallow," "of the order of," "the order of about 5mm," and "substantial portion" were held to be indefinite because the specification lacked some standard for measuring the degrees intended.
In the instant case, claims 86 and 88 are vague and indefinite in the metes and bounds of the phrases “substantially different”, “substantially the same”, “substantially identical”. For example, the term “substantially” is a subjective term that is not defined in the instant disclosure in the context of the claims in any limiting way. Also, the specification of the claimed invention does not define the term “substantially” in any limiting way and there is no standard provided by the instant specification for measuring the degree intended for the term “substantially” as claimed.
Maintained in modified form-Claim Rejections - 35 USC § 101- necessitated by amendments
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.
Claims 82-94, 100-101 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
Step 1: Is the claim to a process, machine, manufacture or composition of matter?
The claims recite a method for assessing effects of a mutation of interest in a cell. Here, because the method is a process, the claims are directed to at least one statutory category of invention (Step 1: YES).
Step 2(A), Prong 1: Does the claim recite an abstract idea, law of nature or natural phenomenon?
Under the broadest reasonable interpretation, the terms of the claim are presumed to have their plain meaning consistent with the specification as it would be interpreted by one of ordinary skill in the art. See MPEP 2111. The claims recite abstract ideas which are mental processes: “a method for assessing effects of a mutation of interest in a cell”:
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These steps of determining effects/ratio/change, correlating, defining, assessing the effect of the mutation on the parameter of interest are mental steps of analyzing data for assessing the effect of the mutation on the parameter of interest. Therefore, the instant claims are reasonably considered as reciting mental steps that are judicial exceptions (Step 2A, Prong I: YES).
Step 2(A), Prong 2: Does the claims recite additional elements that integrate the judicial exception into a practical application?
In view of foregoing analysis, it is apparent that the claims recite judicial exceptions that are abstract ideas which are mental processes. While the claims would still be patent-eligible if “the claims as a whole integrate the recited judicial exceptions (Abstract ideas) into a practical application of the exception”, the additional limitations recited in the instant claims do not integrate the judicial exceptions into a practical application of the recited judicial exceptions. For example, base claim 82 is directed to a method for assessing effects of a mutation of interest in a cell. The recitation of “for assessing effects of a mutation of interest” is an abstract ideas and mental steps that does not indicate any structural or manipulative difference in the invention recited in the body of the claim. Claim 83 specifies “further comprises a step of determining an initial frequency of cells…..” which is judicial exception of mental steps or abstract ideas. Claim 84 specifies “determining a frequency of cells with an indel….” which is judicial exception of mental steps or abstract ideas. Claim 85 specifies “said mutation of interest and/or said synonymous mutation lies within the binding region of said nuclease”. Claim 86 specifies the mixed population also comprises cells in which the mutation of the first or the second oligonucleotides has been introduced, and/or wherein one or more indel mutations have been introduced in the target nucleic acid sequence, and the initial and the subsequent frequency of cells with an indel is further subdivided, and the frequency of cells with an indel is further subdivided. Claim 87 specifies if the initial ratio of cells is lower than the subsequent ratio of cells, the mutation is characterized as a mutation having a positive/negative/no effect on the temporal parameter. Claim 88 specifies step B. v) comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest and wherein: If the ratio of cells in said subpopulation is greater/lower/ substantially the same than in said reference subpopulation, the mutation is characterized as a mutation having a positive effect on the spatial parameter. Claim 89 specifies the first oligonucleotide comprises at least one first mutation, which is a synonymous mutation lying inside the binding region of the nuclease, and further comprises at least one second mutation, which is a non-synonymous mutation of interest lying outside the binding region of the nuclease, and wherein the second oligonucleotide comprises at least one synonymous mutation lying within the same region as the first mutation and further comprises at least one further synonymous mutation lying inside the same region as the second mutation, wherein the first mutation and the synonymous mutation when introduced in the target nucleic acid sequence prevent the nuclease and the targeting means from binding to and/or generating a further SSB or a further DSB in the resulting nucleic acid sequence, preferably wherein the first mutation and the synonymous mutation are identical. Claim 90 specifies the nuclease comprises or consists of a CRISPR/Cas nuclease and the targeting means comprise or consist of a guide RNA capable of hybridizing to the genomic target region. Claim 91 specifies step A v) and/or step B.vi) comprises amplifying a region comprising the target nucleic acid sequence to produce an amplicon comprising the target nucleic acid sequence. Claim 92 specifies the amplifying is performed using primers that anneal outside a region substantially identical or complementary to the first or second oligonucleotides. Claim 93 specifies step B. v) comprises a step of spatially separating the cells to separate the mixed cell population into subpopulations on the basis of a cell marker. Claim 94 specifies step B. v) comprises defining the subpopulations on the basis of a cell property. Claim 100 is directed to a method of assessing the effects of a mutation of interest in a cell comprising: assessing said effects in the population of host cells. Claim 101 is directed to the method comprises the steps according to claim 100.
In summary, there are no additional elements recited in the claims, considered individually or in combination, that integrate the judicial exception of abstract ideas into a practical application of the judicial exception and the claims are therefore considered to be directed to the judicial exceptions. (Step 2A, Prong II: NO).
Step 2(B): Does the claim recite additional elements that amount to significantly more than the judicial exception?
The claims as a whole do not include additional elements that are sufficient to amount to
significantly more than the judicial exception of abstract ideas. In the instant case, the method steps recited in the claims all appear to be data-gathering steps that are all well-understood, routine, and conventional activity in the art:
Findlay et al (Nature 562, 217–222 (2018). doi:10.1038/s41586-018-0461-z) teach saturation genome editing of BRCA1 exons in the human haploid cell line HAP1 (see page 217, right column and Fig. 1a) and stated that “we set out to apply genome editing to measure the functional consequences of all possible SNVs in key regions of BRCA1, regardless of whether they have been previously observed in a human … In each experiment, a single exon is subjected to saturation genome editing (SGE)22, wherein all possible SNVs are simultaneously introduced and concurrently assayed. We used SGE to measure functional effects for 3,893 SNVs, comprising 96.5% of all possible SNVs in the targeted exons” (Page 217, right column, 2nd para.).
Findlay et al teach HDR library design and cloning: Array-synthesized oligonucleotides were designed as follows for each saturation genome editing region (that is, a BRCA1 exon). The sequence to be mutated (~100 bp) was obtained from the human genome (hg19) and a synonymous substitution was introduced at the chosen Cas9 target site (for example, a substitution at the PAM site). This ‘fixed’ substitution in the library was included in design to serve multiple purposes: (i) plasmid library molecules harboring the substitution are predicted to be cleaved less frequently by Cas9–gRNA complexes, (ii) single-nucleotide variants (SNVs) introduced to cells are predicted to be depleted via Cas9 re-cutting less frequently as a consequence of the fixed substitution, and (iii) sequencing reads can be filtered on the fixed substitution to distinguish true SNVs introduced via HDR from sequencing errors. A second synonymous substitution at an alternative CRISPR target site was introduced to the sequence as well, such that the SNV library for each exon would be compatible with multiple gRNAs. Next, a sequence was created for every single nucleotide substitution on this template. For all sequences, adapters were added to both ends to enable PCR amplification from the oligonucleotide pool. For each SGE region, the total number of oligonucleotides designed was three times the length of the region, plus the oligonucleotide template without any SNV (for example, for a 100-bp SGE region, 301 total oligonucleotides were designed) (see first page of the method left column).
Findlay et al teach sequencing and data analysis. Sequencing was performed on an Illumina NextSeq or MiSeq instrument (see second page of the method left column), and stated that “to calculate function scores for each SNV, we first calculated the log2 ratio of the frequency of a SNV on day 11 to its frequency in the plasmid library. Second, positional biases in editing rates were modelled using day 5 SNV frequencies and subtracted (Extended Data Fig. 5). Third, to enable comparisons between exons, we normalized function scores such that the median synonymous and nonsense SNV in each experiment matched global medians. Lastly, a small number of SNVs that could not confidently be scored were filtered out (Extended Data Fig. 6)” (Page 218, right column, 3rd para)
Thus, Findlay et al demonstrate a method for assessing effects of a mutation of interest on various cell parameters in a mixed, non-clonally derived cell population, comprising both the mutation of interest as well as one or more synonymous mutations. Therefore, it is reasonable to conclude based on the available prior art that the claims do not include additional elements that are sufficient to amount to significantly more than the “abstract idea” itself. Therefore, the answer to step 2B of the 101 Subject Matter Eligibility Test is “No” (Step 2B: NO).
Conclusion: Given the analysis above, it is reasonable to conclude that claims 82-94, 100-101 encompass embodiments that are not directed at patent-eligible subject matter.
Response to Arguments
Applicants’ arguments filed 07-16-2026 have been fully considered but they are not persuasive.
1. Applicants argue that “the claims do not recite a mental process under step 2a, prong one… Claim 82 requires physical manipulation of biological material and creation of a non-naturally occurring mixed population of genetically modified cells. In particular, the claim requires introducing a nuclease capable of generating single-strand or double- strand breaks into cells, introducing defined oligonucleotides into those cells, generating genomic modifications by integration or copying of the oligonucleotides into a target nucleic acid sequence, and incubating the resulting mixed population of cells under defined conditions. These are technical laboratory operations that cannot practically be performed in the human mind and that fundamentally alter the state of the cells….” (remarks page 11-12)
Response to Arguments:
As described above, the claims recite several mental step with data gathering for determining effects/ratio/change, correlating, defining, assessing the effect of the mutation on the parameter of interest that are mental steps of analyzing data for assessing the effect of the mutation on the parameter of interest. Therefore, the instant claims are reasonably considered as reciting mental steps that are judicial exceptions.
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2. Applicants argue that “Any Alleged Judicial Exception is Integrated into a Practical Application under Step 2A, Prong Two ….The claimed method defines a specific experimental assay architecture in which a mutation of interest and a synonymous mutation are introduced into the same cell population, and the effect of the mutation of interest is assessed by measuring relative frequencies within that mixed population over time and/or across spatially defined subpopulations. This provides a concrete technical solution to a technical problem in the fields of genome editing and functional genomics: reliably assessing mutation effects while controlling for variability in editing efficiency, cell growth, and experimental conditions…” (Remarks, page 12-13).
Response to Arguments:
It is noted that the claims are directed to “a method for assessing effects of a mutation of interest in a cell”. The identity of the “mutation of interest” as well as “effects” of this mutation are within the scope of mental steps which is also the final goal of the method. The claims recite various mental steps that are not integrated into a practical application under step 2a, prong two such as “assessing effects of a mutation of interest in a cell”, “determining the effect of the mutation of interest”, “determining an initial ratio of cells in which the mutation of interest”, “determining a subsequent ratio of cells”, “determining a change in ratio between the initial ratio and the subsequent ratio”, “correlating said change in ratio to the parameter of interest”, “defining and/or spatially separating subpopulations of cells on a basis of said spatial parameter of interest in each subpopulation”, “determining, for each subpopulation, a ratio of cells”, “correlating said ratio to the measured spatial parameter of interest for each subpopulation”, “assessing the effect of the mutation on the parameter of interest”.
Thus, the claims are directed to gathering and analyzing data. They are comprising mental steps of assessing mutation effects of genome editing and functional genomics. The mental steps are in a genome-editing-based functional assay that are not integrated into a practical application under step 2a, prong two.
3. Applicants argue that “the claims recite significantly more under step 2b …..The claimed steps are not generic data gathering or insignificant post-solution activity. Rather, the claims define a specific and structured experimental protocol involving defined molecular reagents, defined genetic modifications, a mixed population of genetically modified cells, incubation under defined conditions, and defined modes of experimental analysis. These limitations, individually and as an ordered combination, amount to significantly more than any alleged abstract idea” (remarks, page 13-14).
Response to Arguments:
As mentioned above, the method steps recited in the claims all appear to be data-gathering steps that are all well-understood, routine, and conventional activity in the art: Findlay et al demonstrate a method for assessing effects of a mutation of interest on various cell parameters in a mixed, non-clonally derived cell population, comprising both the mutation of interest as well as one or more synonymous mutations. Therefore, it is reasonable to conclude based on the available prior art that the claims do not include additional elements that are sufficient to amount to significantly more than the “abstract idea” itself. Therefore, the answer to step 2B of the 101 Subject Matter Eligibility Test is “No”.
Maintained in modified form - Claim Rejections - 35 USC § 103- necessitated by amendments
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.
Claims 82-86, 90-92, 100-101 are rejected under 35 U.S.C. 103 as being unpatentable over Findlay et al (Nature 562, 217–222 (2018). doi:10.1038/s41586-018-0461-z).
Regarding to claim 82 and 100 and 101, Findlay et al teach “we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression…” (Abstract) (For the preamble).
Findlay et al teach “Many genes in the HDR pathway, including the hereditary cancer predisposition genes BRCA1, BRCA2, PALB2 and BARD16, have been deemed essential in the human haploid cell line HAP123 (Fig. 1a). To confirm this, we transfected HAP1 cells with a plasmid co-expressing Cas9 and guide RNAs (gRNAs) targeting each of these genes” (Page 217, right column, last para.). Findlay et al teach “ S. pyogenes Cas9 target sites were chosen for SGE experiments on multiple criteria, assessed in the following order: (i) to induce cleavage within BRCA1 coding sequence, (ii) to target a genomic site permissive to synonymous substitution within the guanine dinucleotide of the PAM or the protospacer, (iii) to have minimal predicted off target activity, (iv) to have maximal predicted on-target activity” (first page of the Method, 3rd para. “gRNA design and cloning”). Findlay et al teach HDR library design and cloning: Array-synthesized oligonucleotides were designed as follows for each saturation genome editing region (that is, a BRCA1 exon). The sequence to be mutated (~100 bp) was obtained from the human genome (hg19) and a synonymous substitution was introduced at the chosen Cas9 target site (for example, a substitution at the PAM site). This ‘fixed’ substitution in the library was included in design to serve multiple purposes: (i) plasmid library molecules harboring the substitution are predicted to be cleaved less frequently by Cas9–gRNA complexes, (ii) single-nucleotide variants (SNVs) introduced to cells are predicted to be depleted via Cas9 re-cutting less frequently as a consequence of the fixed substitution, and (iii) sequencing reads can be filtered on the fixed substitution to distinguish true SNVs introduced via HDR from sequencing errors. A second synonymous substitution at an alternative CRISPR target site was introduced to the sequence as well (see first page of the method left column, 5th para.). (For claim 82 items i), ii)- a), b), c) and claim 100 items i), ii), iii) and claim 101 items i), ii)- a), b), c) ).
Findlay et al teach “Transfection of HAP1 cells …. For each SGE transfection, 10 million cells were passaged to a 10-cm dish. The next day (day 0), cells were co-transfected with 12 μg of the Cas9/gRNA plasmid (pX459) and 3 μg of the SNV library corresponding to a single exon. Negative control transfections were performed for each library using a pX459 vector targeting HPRT1 instead of BRCA1, thus preventing genomic integration of the library.” (see the method, page 1, right column, 4th para.). Note: Findlay et al teach “…..S. pyogenes Cas9 target sites were chosen for SGE experiments on multiple criteria, assessed in the following order: (i) to induce cleavage within BRCA1 coding sequence, (ii) to target a genomic site permissive to synonymous substitution within the guanine dinucleotide of the PAM or the protospacer, (iii) to have minimal predicted off target activity43, (iv) to have maximal predicted on-target activity…...” (see the method, page 1, left column, 3rd para.) (For claim 82 and 101 the claimed: obtaining a mixed population of cells comprising cells with no mutation, mutation of interest, synonymous mutation).
Findlay et al measure changes in variant abundance during cell growth which represents a temporal change in cell fitness associated with each variant: “A population of 20 million HAP1 cells was co-transfected on day 0 with a corresponding SNV library and Cas9/gRNA plasmid. Variant frequencies were quantified by targeted sequencing of the edited exon from genomic DNA (gDNA) collected on day 5 and day 11” (Page 218, left column, 2nd para.). Findlay et al teach “Function scores for 3,893 BRCA1 SNVs: To calculate function scores for each SNV, we first calculated the log2 ratio of the frequency of a SNV on day 11 to its frequency in the plasmid library. Second, positional biases in editing rates were modelled using day 5 SNV frequencies and subtracted (Extended Data Fig. 5). Third, to enable comparisons between exons, we normalized function scores such that the median synonymous and nonsense SNV in each experiment matched global medians. Lastly, a small number of SNVs that could not confidently be scored were filtered out (Extended Data Fig. 6). Altogether, we obtained function scores for 3,893 SNVs, which comprise 96.5% of all possible SNVs within or immediately intronic to these exons (Supplementary Table 1; https://sge.gs.washington.edu/ BRCA1/).”(Page 218, right column, 3rd para.). Since Findlay et al calculated and normalized function scores such that the median synonymous and nonsense SNV in each experiment matched global medians, it is indicating that calculating ratio of the frequency the frequency of variant and normalizing with synonymous mutations was recognized in the prior art to be a result-effective variable, and a person of ordinary skill in art would have been able and motivated to perform the determining a change in ratio during cell growth a plurality of times out of the course of routine optimization (For claim 82, items iii), iv), A-v) and vi) and claim 101) items iii), iv), A-v) and vi).
It is noted that the claims 82 and 101 recite the term “and/or” between items A (temporal parameter) and B (spatial parameter). Thus, either A or B can satisfy the claims.
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to use the teachings of Findlay et al to obtain a mixed population of cells with no mutation, mutation of interest, synonymous mutation and analyze the ratios of frequencies of mutation of interest during cell growth over time as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Findlay et al stated that “Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression. We predict that these results will be immediately useful for the clinical interpretation of BRCA1 variants, and that this approach can be extended to overcome the challenge of variants of uncertain significance in additional clinically actionable genes.” (Abstract) and “we show that SGE is a viable strategy for functionally classifying thousands of variants in a clinically actionable gene, most of which have yet to be observed in a human. We anticipate function scores will prove valuable, both for adjudicating hundreds of observed BRCA1 variants for which the interpretation is currently ambiguous, as well as for providing immediate functional assessments for newly observed variants. This work may also serve as a blueprint for the comprehensive functional analysis of all potential SNVs in clinically actionable genes” (Page 221, right column, last para.). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Findlay et al were successful in assessing effects of large amount of mutations/variants of BRCA1.
Regarding to claim 83-84 and 86, Findlay et al teach determining frequency of cells with an indel in the target nucleic acid sequence which is different from the mutation of the first and the second oligonucleotides during cell growth (day 11 over day 5): “Extended Data Fig. 2 | Analysis of Cas9-induced indels observed in BRCA1 SGE experiments. Variants observed in gDNA sequencing were included in this analysis if (i) they aligned to the reference with either a single insertion or deletion within 15 bp of the predicted Cas9 cleavage site and (ii) were observed at a frequency greater than 1 in 10,000 reads in both replicates …. a, Histograms show the number of unique indels observed of each size …. b, Day 11 over day 5 indel frequencies were normalized to the median synonymous SNV in each replicate and then averaged across replicates to measure selection on each indel. The distribution of selective effects is shown for each experiment as a histogram, in which indels are colored by whether their size was divisible by 3 (that is, ‘in-frame’ versus ‘frameshifting’). Whereas frameshifting variants were consistently depleted, some exons were tolerant to in-frame indels (see Extended Data Fig. 2).
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Note: Findlay et al teach determining the frequencies of mutation of interest (indel) using temporal parameter (Day 11 over day 5) correlating with change in ratio (normalized to the median synonymous SNV).
Regarding to claim 85, Findlay et al teach that the sequence to be mutated (~100 bp) was obtained from the human genome (hg19) and a synonymous substitution was introduced at the chosen Cas9 target site (for example, a substitution at the PAM site) (see first page of the method left column, 5th para.).
Regarding to claim 90, Findlay et al teach “cancer predisposition genes BRCA1, BRCA2, PALB2 and BARD16, have been deemed essential in the human haploid cell line HAP123 (Fig. 1a). To confirm this, we transfected HAP1 cells with a plasmid co-expressing Cas9 and guide RNAs (gRNAs) targeting each of these genes” (Page 217, right column, last para.).
Regarding to claims 91-92, Findlay et al teach “Homology arms were cloned into pUC19 by PCR-amplifying (Kapa HiFi) regions surrounding each targeted exon from HAP1 gDNA. Primers for these reactions were designed such that homology arms would be between 600 bp and 1,000 bp on both sides of the targeted region. Adapters homologous to pUC19 were added to primers to facilitate NEBuilder HiFi Assembly cloning (NEB) into a linearized pUC19 vector. ….. homology arm plasmids were linearized via PCR using primers that conferred 15–20 bp of terminal overlap with the adapter sequences flanking each PCR-amplified oligonucleotide pool.” (First page of the method, left column, last 2 para.).
Response to Arguments
Applicants’ arguments filed 07-16-2026 have been fully considered but they are not persuasive.
1. Applicants argue that
“Findlay fails to teach or suggest the claimed ratio-based assessment strategy using a limited number of predetermined mutations of interest and a synonymous internal reference mutation ….”
“Findlay does not determine the effect of a selected mutation of interest by directly comparing the frequency of cells carrying that mutation to the frequency of cells carrying a synonymous control mutation introduced in parallel into the same population. Instead, Findlay infers functional effects from changes in variant representation in a pooled library, including comparisons to the input plasmid library and library-wide corrections for positional bias in knockin efficiency ….”
“….As a result, the final 'function score' does not correspond to a direct, experimentally measured change over time, but to a model-corrected value combining late time point data, library input data, and an estimated positional correction, which introduces additional uncertainty into the analysis. …. Findlay therefore relies on global normalization and model-based correction across the variant library, rather than on the direct internal-reference comparison as claimed…..”
“In contrast, the instantly claimed methods require determining the effect of a mutation of interest by monitoring the relative frequencies of cells carrying the mutation of interest versus cells carrying a synonymous mutation, including by determining an initial ratio and a subsequent ratio and determining a change in that ratio, or by determining such a ratio in spatially defined subpopulations. Findlay does not teach or suggest this claimed ratio-based assessment strategy”
(Remarks, page 14-15)
Response to Arguments:
Findlay teach ratio-based assessment strategy for mutations of interest and a synonymous internal reference mutation, an initial ratio and a subsequent ratio and determining a change in that ratio:
Findlay et al teach “we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1” (Abstract), and “Function scores for 3,893 BRCA1 SNVs: To calculate function scores for each SNV, we first calculated the log2 ratio of the frequency of a SNV on day 11 to its frequency in the plasmid library. Second, positional biases in editing rates were modelled using day 5 SNV frequencies and subtracted (Extended Data Fig. 5). Third, to enable comparisons between exons, we normalized function scores such that the median synonymous and nonsense SNV in each experiment matched global medians. Lastly, a small number of SNVs that could not confidently be scored were filtered out (Extended Data Fig. 6). Altogether, we obtained function scores for 3,893 SNVs, which comprise 96.5% of all possible SNVs within or immediately intronic to these exons (Supplementary Table 1; https://sge.gs.washington.edu/ BRCA1/).”(Page 218, right column, 3rd para.). Thus, it is indicating that calculating ratio of the frequency the frequency of variant and normalizing with synonymous mutations was recognized in the prior art to be a result-effective variable. Since Findlay et al calculated and normalized function scores such that the median synonymous and nonsense SNV in each experiment matched global medians, a person of ordinary skill in art would have been able and motivated to perform the determining a change in ratio during cell growth a plurality of times out of the course of routine optimization.
Findlay et al teach “ S. pyogenes Cas9 target sites were chosen for SGE experiments on multiple criteria, assessed in the following order: (i) to induce cleavage within BRCA1 coding sequence, (ii) to target a genomic site permissive to synonymous substitution within the guanine dinucleotide of the PAM or the protospacer, (iii) to have minimal predicted off target activity, (iv) to have maximal predicted on-target activity” (first page of the Method, 3rd para. “gRNA design and cloning”).
Applicants argue that “Findlay does not determine the effect of a selected mutation of interest by directly comparing the frequency of cells carrying that mutation to the frequency of cells carrying a synonymous control mutation introduced in parallel into the same population. Instead, Findlay infers functional effects from changes in variant representation in a pooled library, including comparisons to the input plasmid library and library-wide corrections for positional bias in knock in efficiency ….”. This is not persuasive because applicants’ arguments are not commensurate to the scope of the claims:
It is noted that the claims do not require “comparing the frequency of cells carrying that mutation to the frequency of cells carrying a synonymous control mutation” as in applicant arguments. The claims require “correlating said change in ratio to the parameter of interest” (e.g., claim 82 A-vi). In the instant case, Findlay et al teach Function scores for 3,893 BRCA1 SNVs via calculating the log2 ratio of the frequency of a SNV on day 11 and day 5 and normalized function scores such that the median synonymous and nonsense SNV in each experiment matched global medians. (Page 218, right column, 3rd para.). Thus, Findlay et al teach correlating change in ratio to the parameter of interest.
Applicants argue that Findlay infers functional effects from changes in variant representation in a pooled library. It is noted that the claims require “obtaining a mixed population of cells comprising cells in which the mutation of the first or the second oligonucleotides has not been introduced, cells in which only the mutation of the first oligonucleotide has been introduced, and cells in which only the mutation of the second oligonucleotide has been introduced”. Findlay et al also teach obtaining a mixed population of HAP1 cells generated from the pooled library that is comprising cells with no mutation, mutation of interest, synonymous mutation: “HDR library design and cloning ……The sequence to be mutated (~100 bp) was obtained from the human genome (hg19) and a synonymous substitution was introduced at the chosen Cas9 target site (for example, a substitution at the PAM site) ……. To construct the HDR library, homology arm plasmids were linearized via PCR using primers that conferred 15–20 bp of terminal overlap with the adapter sequences flanking each PCR-amplified oligonucleotide pool…..” (see the method, page 1, left column, “HDR library design and cloning”) and transfecting the HDR library to generate a mixed population of cells: “Transfection of HAP1 cells …. For each SGE transfection, 10 million cells were passaged to a 10-cm dish. ….. Negative control transfections were performed for each library using a pX459 vector targeting HPRT1 instead of BRCA1, thus preventing genomic integration of the library……” (see the method, page 1, right column, 4th para.).
Applicants argue that “As a result, the final 'function score' does not correspond to a direct, experimentally measured change over time, but to a model-corrected value combining late time point data, library input data, and an estimated positional correction, which introduces additional uncertainty into the analysis”. This is not persuasive because it is noted that the claims only require “ iv) determining an effect of the mutation of interest on the parameter of interest” under condition of item A (a temporal parameter) or B (a spatial parameter) and “vi) correlating said change in ratio to the parameter of interest” (e.g., claim 82 A-vi). In the instant case: Findlay et al teach a temporal parameter frequency: determining the frequencies of mutation of interest (indel) using temporal parameter (Day 11 over day 5) correlating with change in ratio (normalized to the median synonymous SNV):
Findlay et al teach determining frequency of cells with an indel in the target nucleic acid sequence during cell growth (day 11 over day 5): “Extended Data Fig. 2 | Analysis of Cas9-induced indels observed in BRCA1 SGE experiments. Variants observed in gDNA sequencing were included in this analysis if (i) they aligned to the reference with either a single insertion or deletion within 15 bp of the predicted Cas9 cleavage site and (ii) were observed at a frequency greater than 1 in 10,000 reads in both replicates …. a, Histograms show the number of unique indels observed of each size …. b, Day 11 over day 5 indel frequencies were normalized to the median synonymous SNV in each replicate and then averaged across replicates to measure selection on each indel. The distribution of selective effects is shown for each experiment as a histogram, in which indels are colored by whether their size was divisible by 3 (that is, in-frame’ versus ‘frameshifting’). Whereas frameshifting variants were consistently depleted, some exons were tolerant to in-frame indels (see Extended Data Fig. 2).
2. Applicants argue that
“Findlay also does not teach or suggest a true comparison between an early and a late time point for a defined mutation pair in the sense of the present claims. Although sequencing is performed at multiple time points, the analysis in Findlay focuses on library-wide depletion patterns and model-based function scores, rather than on a direct, quantitative comparison of mutation frequencies for a specific mutation of interest relative to a synonymous control within the same population. Although Findlay performs sequencing at multiple time points, its analysis focuses on library-wide depletion patterns and model-based function scores. Specifically, although Findlay sequences genomic DNA at day 5 and at day 11, the day 5 measurement is used as a technical correction input (as basis of modelling) rather than as an initial functional read out for a defined mutation relative to a defined internal comparator ….. Thus, Findlay does not teach determining an initial ratio and a subsequent ratio between cells carrying a mutation of interest and cells carrying a synonymous mutation and then using the change in that ratio as the functional readout ….. ”
“Findlay also fails to teach or suggest determining the effect of a mutation of interest by comparing its frequency to the frequency of a synonymous control mutation introduced in parallel into the same population and tracking a change in that ratio from an initial to a subsequent time point”
“The instantly claimed methods can also be distinguished from Findlay because the methods are directed to quantitative assessment of selected mutations rather than saturation scale screening”
(Remarks, page 16-17)
Response to Arguments:
Findlay et al do teach or suggest a true comparison between an early and a late time point:
Findlay et al do teach Extended Data Fig. 2b, Day 11 over day 5 indel frequencies were normalized to the median synonymous SNV in each replicate and then averaged across replicates to measure selection on each indel. The distribution of selective effects is shown for each experiment as a histogram, in which indels are colored by whether their size was divisible by 3 (that is, in-frame’ versus ‘frameshifting’). Whereas frameshifting variants were consistently depleted, some exons were tolerant to in-frame indels (see Extended Data Fig. 2).
Applicants argue that Findlay et al do not teach quantitative comparison of mutation frequencies for a specific mutation of interest relative to a synonymous control within the same population. Findlay et al do not teach determining the effect of a mutation of interest by comparing its frequency to the frequency of a synonymous control mutation. However, it is not persuasive because
The claims do not specify the “specific mutation” nor “quantitative comparison” nor “comparing its frequency”. The claims require “…. measuring absolute frequencies of introduction of the mutation of interest and the synonymous mutation in the cell population, and determining an initial ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced ….. vi) correlating said change in ratio to the parameter of interest” (see claim 82, A-v) and vi)). In the instant case, Findlay et al teach Day 11 over day 5 indel frequencies were normalized to the median synonymous SNV in each replicate and then averaged across replicates to measure selection on each indel (see Extended Data Fig. 2). Thus, applicants’ arguments are not commensurate to the scope of the claims.
3. Applicants argue that
“Findlay fails to teach or suggest the spatial-parameter embodiment recited in the instant claims. Findlay uses bulk sampling of a pooled edited cell population followed by sequencing-based quantification of variant abundances and computational processing to derive library-wide function scores. It contains no teaching of defining or spatially separating subpopulations of cells based on a spatial parameter, nor of correlating the ratio between cells carrying a mutation of interest and cells carrying a synonymous mutation with such a spatial parameter”
(Remark, page 18)
Response to Arguments:
It is noted that the claims 82 and 101 recite the term “and/or” between items A (temporal parameter) and B (spatial parameter). Thus, either items A (temporal parameter) or B (spatial parameter) can satisfy the claims.
Conclusion
No claim is allowed.
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.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
