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 filed on 01-05-2026 have been received and entered. Claims 1-81 have been canceled. Claims 82-101 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 traversal is on the ground(s) that “The asserted common technical feature is directed to assessing the effects of mutations 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, while ensuring the results are statistically significant ….. The change in ratio over time between the cells having introduced either the mutation of interest or the synonymous mutation, or the specific ratio measured in specific, spatially defined subpopulations, is then used to measure the effect of the mutation of interest on a cell parameter of interest”.
This is not found persuasive because the special feature and group II are directed to product by process claim. MPEP 2113 states "[E]ven though product by process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product by process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695,698,227 USPQ 964, 966 (Fed. Cir. 1985).
The product in base claim 95 of group II require: (i) a nuclease or a polynucleotide encoding said nuclease; (ii) a first oligonucleotide comprising a mutation of interest; (iii) a second oligonucleotide comprising a synonymous mutation. The cited Zhang et al reference in the preceding office action teaches the SF3B1 K700E mutation (or synonymous mutations for the WT control) was knocked into K562 cells using CRISPR/Cas9 technology with the human SF3B1 CRISPR guide RNA and the single-stranded oligodeoxynucleotides (ssODNs, Integrated DNA Technologies) for WT (See page e5 of the Method Details). It is noted that Cas9 is a nuclease capable of generating one or more single-stranded breaks (SSBs) or double-strand breaks (DSBs) in the target nucleic acid sequence. Zhang et al stated that “to identify proteins differentially associated with mutant and WT SF3B1, we wished to perform affinity purification using cells expressing either K700E or WT SF3B1 with affinity tags. To this end, we engineered K562 myelogenous leukemia cells using CRISPR/Cas9 technology to knock in the hotspot K700E mutation (or synonymous mutations for the WT control), followed by addition of mono-allelic His6 (hexahistidine)-FLAG tandem tags at the N terminus of the knockin SF3B1 allele (see STAR Methods). Using RT-PCR, we confirmed that cells expressing the tagged K700E SF3B1 recapitulated the 30ss switch (Figure 2A)” (Page 85, right column).
The requirement is still deemed proper and is therefore made FINAL.
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.
Claims 82-94, 100-101 are under consideration.
Priority
This application is a 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.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 08-08-2023 are in compliance with the provisions of 37 CPR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
Claim Objections
Claims 87-88, 100 and 101 are objected to because of the following informalities:
Claims 87-88 are objected because of including a capitalized “If” at the start of each ‘if this, then that’ limitations recited in the claims. Appropriate correction is required.
Claim 100 depends from withdrawn claim 95. For the sake of compact prosecution, the claim 100 is interpreted to read with limitations of the product of claim 95. Appropriate correction is required.
Claim 101 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n). Accordingly, the claim 101 not been further treated on the merits. 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 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.
Claim Rejections - 35 USC § 112
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, 87, 88, 89, 92 are 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, 87, 88, 92 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, 87, 88, 92 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.
Claim 89 recite the term “preferably” which is vague and render the claims indefinite. It is unclear whether the phrase after the term “preferably” is a required limitation for the claim or not. It is also unclear the circumstances where the limitations recited after “preferably” would be the “preferred” limitation. Appropriate correction/clarification is required.
Claim Rejections - 35 USC § 101
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 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, calculating, 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 82.
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 to patent-eligible subject matter.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 82-86, 90-92, 100 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 , 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” (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 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.” (For 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.). 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. A person of ordinary skill in the art would have been motivated to perform the determining a change in ratio during cell growth a plurality of times out of the course of routine optimization (For items iii), iv), A-v) and vi) ).
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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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.)
Conclusion
No claim is allowed.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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