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 .
2. This action is in response to the papers filed February 11, 2026. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any new grounds of rejection presented in this Office Action are necessitated by Applicant's amendments. Any rejections or objections not reiterated herein have been withdrawn. This action is made FINAL.
Claims 13, 25, 66-68, 70, 72-77, 79, and 82-95 are currently pending and have been examined herein.
Claim Rejections - 35 USC § 101
3. 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 13, 25, 66-68, 70, 72-77, 79, and 82-95 are rejected under 35 U.S.C. 101 because the claimed invention is directed to judicial exception without significantly more. The claims recite a judicial exception that is not integrated into a practical application. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception. The claim analysis is set forth below.
Step 1: The claims are directed to the statutory category of a process.
Step 2A, prong one: Evaluate Whether the Claim Recites a Judicial Exception
The instant claims recite abstract ideas.
The claims recite a step of “calculating” a mutant frequency for a plurality of the DNA fragments by calculating the number of unique mutations per duplex base-pair sequenced (clm 13). Mathematical concepts, such as mathematical calculations, are considered to be abstract ideas.
The claims recite a step of “determining” a mutation pattern for the plurality of the DNA fragments, wherein the mutation pattern includes mutation type, mutation trinucleotide context, and genomic distribution of mutations (clm 13). The “determining” step broadly encompasses an activity that can be performed in the human mind. While some types of pattern determination require sophisticated software and are so complex they cannot be performed in the human mind, the claims do not require this. The “determining” could be performed by reading a laboratory report and thinking about the mutation type, mutation trinucleotide context, and genomic distribution of mutations.
The claims recite a step of “comparing” the mutation signature of the test agent with mutation signatures of one or more known genotoxins (clm 66). The “comparing” step broadly encompasses an activity that can be performed in the human mind. While some types of comparisons require sophisticated software and are so complex they cannot be performed in the human mind, the claims do not require this. The “comparing” could be performed by reading a laboratory report and thinking about the mutation signatures of the test agent and genotoxin.
The claims recite that the mutagenic signature is generated by computational pattern matching (clm 70). The specification (para 0084) states “In an embodiment, the mutation spectrum is generated by computational pattern matching (e.g., unsupervised hierarchical mutation spectrum clustering)”. It is noted that hierarchical clustering is an unsupervised learning method for clustering data points. The algorithm builds clusters by measuring the dissimilarities between data. Algorithms are mathematical concepts and thus considered to be abstract ideas.
The claims recite a step of “comparing” the first strand sequence read and the second strand sequence read (clm 25). The “comparing” step broadly encompasses an activity that can be performed in the human mind. While some types of comparisons require sophisticated software and are so complex they cannot be performed in the human mind, the claims do not require this. The “comparing” could be performed by reading a sequence alignment.
The claims recite a step of “determining” a mutation signature of the test agent by analyzing sequencing reads (clm 25). The “determining” step broadly encompasses an activity that can be performed in the human mind. While some types of mutation signatures require sophisticated software and are so complex they cannot be performed in the human mind, the claims do not require this. The “determining” could be performed by reading a laboratory report and thinking about the mutation type or genomic distribution of mutations.
The claims recite a step of “comparing” the mutation signature of the test agent to the mutation signature of known genotoxins (clm 25). The “comparing” step broadly encompasses an activity that can be performed in the human mind. While some types of comparisons require sophisticated software and are so complex they cannot be performed in the human mind, the claims do not require this. The “comparing” could be performed by reading a laboratory report and thinking about the mutation signatures of the test agent and genotoxin.
The claims recite a step of “assessing” if the mutant frequency, mutation type or mutation type threshold is above a safe threshold level (clm 25). The “assessing” step broadly encompasses an activity that can be performed in the human mind. The “assessing” could be performed by thinking about the levels.
The claims recite a step of “determining” if the mutant frequency exceeds a safe threshold level (clm 25). The “determining” step broadly encompasses an activity that can be performed in the human mind. The “determining” could be performed by thinking about the levels.
Step 2A, prong two: Evaluate Whether the Judicial Exception Is Integrated Into a Practical Application
The claims do NOT recite additional steps or elements that integrate the recited judicial exceptions into a practical application of the exception(s). For example, the claims do not practically apply the judicial exception by including one or more additional elements that the courts have stated integrate the exception into a practical application:
An additional element reflects an improvement in the functioning of a computer, or an improvement to other technology or technical field;
An additional element that applies or uses a judicial exception to effect a particular treatment or prophylaxis for a disease or medical condition;
An additional element implements a judicial exception with, or uses a judicial exception in conjunction with, a particular machine or manufacture that is integral to the claim;
An additional element effects a transformation or reduction of a particular article to a different state or thing; and
An additional element applies or uses the judicial exception in some other meaningful way beyond generally linking the use of the judicial exception to a particular technological
environment, such that the claim as a whole is more than a drafting effort designed to monopolize the exception.
In addition to the judicial exceptions, claim 13 recites duplex sequencing DNA fragments extracted from a test subject exposed to the test agent. In addition to the judicial exceptions, claim 25 recites steps of preparing a sequencing library from a sample comprising a plurality of double-stranded DNA fragments from a biological source exposed to the test agent, wherein preparing the sequence library comprises ligating asymmetric adapter molecules to the plurality of double-stranded DNA fragments to generate a plurality of adapter-DNA molecules and (b) sequencing first and second strands of the adapter-DNA molecules to provide a first strand sequence read and a second strand sequence read for at least a portion of the adapter-DNA molecules. These steps are not considered to integrate the judicial exception into a practical application because they merely add insignificant extra-solution activity (data gathering) to the judicial exception.
Step 2B: Evaluate Whether the Claim Provides an Inventive Concept
In addition to the judicial exceptions, claim 13 recites duplex sequencing DNA fragments extracted from a test subject exposed to the test agent. In addition to the judicial exceptions, claim 25 recites steps of preparing a sequencing library from a sample comprising a plurality of double-stranded DNA fragments from a biological source exposed to the test agent, wherein preparing the sequence library comprises ligating asymmetric adapter molecules to the plurality of double-stranded DNA fragments to generate a plurality of adapter-DNA molecules and (b) sequencing first and second strands of the adapter-DNA molecules to provide a first strand sequence read and a second strand sequence read for at least a portion of the adapter-DNA molecules. These steps do not amount to significantly more because they simply append well understood, routine, and conventional activities previously known in the art, specified at a high level of generality, to the judicial exceptions.
The prior art also demonstrates the well understood, routine, conventional nature of additional elements because it teaches that duplex consensus sequencing methods where known in the art (see discussion of Schmitt (US 2015/0044687 Pub 2/12/2015) below).
Further it is noted that the courts have recognized the following laboratory techniques as well-understood, routine, conventional activity in the life science arts when they are claimed in a merely generic manner (e.g., at a high level of generality) or as insignificant extra-solution activity.
Determining the level of a biomarker in blood by any means, Mayo, 566 U.S. at 79, 101 USPQ2d at 1968; Cleveland Clinic Foundation v. True Health Diagnostics, LLC, 859 F.3d 1352, 1362, 123 USPQ2d 1081, 1088 (Fed. Cir. 2017);
Using polymerase chain reaction to amplify and detect DNA, Genetic Techs. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016); Ariosa Diagnostics, Inc. v. Sequenom, Inc., 788 F.3d 1371, 1377, 115 USPQ2d 1152, 1157 (Fed. Cir. 2015);
Detecting DNA or enzymes in a sample, Sequenom, 788 F.3d at 1377-78, 115 USPQ2d at 1157); Cleveland Clinic Foundation 859 F.3d at 1362, 123 USPQ2d at 1088 (Fed. Cir. 2017);
Analyzing DNA to provide sequence information or detect allelic variants, Genetic Techs., 818 F.3d at 1377; 118 USPQ2d at 1546;
Amplifying and sequencing nucleic acid sequences, University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 764, 113 USPQ2d 1241, 1247 (Fed. Cir. 2014)
For the reasons set forth above the claims are not directed to patent eligible subject matter.
Response To Arguments
4. In the response the Applicants traversed the rejection under 35 USC 101. The Applicants argue that the current claims provide methods for generating a mutation signature or assessing the genotoxic potential of a test agent. As explained in the present application, the present claims allow for cost- and time-efficient detection of genotoxic effects of potential genotoxic agents with high accuracy and sensitivity. They argue that the present claims have many applications, including "assessing cancer risk, identifying carcinogens and predicting the impact of exposure in humans." They argue that the claimed methods "allow direct measurement of agent-induced mutations in any genomic context of any organism, and without need for clonal selection," in comparison with existing methods that are "slow, cumbersome, and/or limited in the information that they provide." Id., 0157, 0159. Accordingly, a skilled artisan reading the present application would recognize that the present claims can greatly improve fields such as medical diagnosis and treatment (e.g., cancer), and environmental pollution testing.
This argument has been reviewed but is not persuasive. Herein the “technology” used by the claim is duplex DNA sequencing. The additional elements cited by the Applicants, do not improve the technology of duplex DNA sequencing and do not improve any other technology. The additional elements cited by the Applicants, do not make the technology of duplex DNA sequencing work better and do not make any other technology work better. Therefore, there is no improvement nor is the exception integrated into a practical application.
Further the Applicants argue that the references of record do not disclose nor suggest all of the features of the claimed invention. Accordingly, the amended claims recite additional elements such that the claim as a whole amounts to significantly more than the judicial exception.
This argument has been fully considered but is not persuasive. Applicants are reminded that the test for patent eligibility is distinct from the analysis of whether claims are anticipated or obvious in view of the prior art. The rejection is maintained.
Claim Rejections - 35 USC § 103
5. 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.
6. Claims 13, 67-68, 72, and 75 are rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Manjanatha (Environmental and Molecular Mutagenesis 56:446-456 (2015), Schmitt (US 2015/0044687 Pub 2/12/2015), and Alexandrov (Nature 500 415-421 2013).
Regarding Claim 13 Matsumura teaches a method for evaluating genotoxicity of a substance. The method comprises (1) obtaining DNAs from a test group, the test group is a cell population exposed to the test substance; (2) sequencing fragments of the DNAs to obtain one or more read sequences per fragment; (3) comparing each of the one or more read sequences with a reference sequence to detect sites of mismatch bases between the each read sequence and the reference sequence, wherein the reference sequence is a known sequence in the DNAs; (4) obtaining the sites detected in the step (3) as sites of mutation; (5) classifying each of the obtained mutations according to mutation patterns; and (6) determining respective mutation frequencies of the mutation patterns obtained in the step (5) (para 0017). Matsumura teaches obtaining information on a mutation at the mutation site. Examples of the information to be obtained include, but are not limited to, the type of the mismatched site (mutation site) (e.g., a substitution site, a deletion site or an insertion site), the type of the base at the site and the type of a base (or a base pair) before the mutation (e.g., the type of a base at a position corresponding to the site on the reference sequence), and the types of both adjacent bases of the site (e.g., the types of both bases adjacent to the position corresponding to the site on the reference sequence) (para 54). Matsumura further teaches that that the obtained pieces of information can be collected to create a database for mutation analysis. For example, a database of all information on the mutation site obtained from each read sequence may be created; a database may be created in which mutation information obtained from each read sequence is classified by type of the mutation site; a database may be created in which mutation information obtained from each read sequence is classified by type of a base before the mutation (e.g., in the reference sequence) or after the mutation at the mutation site; a database may be created in which mutation information obtained from each read sequence is classified by base length of the mutation site (e.g., length of an insertion, deletion or substitution site); or a database may be created by combining these classifications. Alternatively, a database may be created together with information on whether the identified position of the mutation site on the genome corresponds to a coding region or a noncoding region of a gene and, if the mutation site is in the coding region, information on whether the coding region is an intron or an exon or resides in a strand to be transcribed into RNA or not (para 0056). Thus Matsumura teaches a method for generating a mutation signature of a test agent, comprising: sequencing DNA fragments extracted from a cell population exposed to the test agent; and generating a mutagenic signature of the test agent, comprising: calculating a mutant frequency for a plurality of the DNA fragments by calculating the number of unique mutations per duplex base-pair sequenced; and determining a mutation pattern for the plurality of the DNA fragments, wherein the mutation pattern includes mutation type, mutation trinucleotide context, and genomic distribution of mutations.
Regarding Claim 68 the prior art of Matsumura teaches the analysis of human cells grown in culture (para 0043).
Matsumura does not teach a method wherein the DNA fragments are extracted from a sample obtained from a subject that has been exposed to the test agent 30 days or less prior to the sample being obtained from the subject (clm 13). Matsumura does not teach a method wherein the mutation signature of the test agent varies on the type of sample (clm 67). Matsumura does not teach a method wherein the subject is a transgenic animal, and wherein the DNA fragments comprise one or more transgene (clm 75).
However Manjanatha teaches that to investigate whether or not gene mutation is involved in the etiology of acrylamide (AA) or glycidamide (GA) induced mouse lung carcinogenicity, we screened for cll mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19–111 mg/kg bw/days). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P 0.01). The predominant types of mutations in the lung cll gene from AA- and GA treated mice were A:T → T:A, and G:C →C:G transversions, and -1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted (abstract). Further Fig 1 compares the cll mutation spectra between lung, liver, and testis from unexposed BB mice and BB mice exposed to 7.0 nM AA or GA. There were significant differences in the mutational spectra between lung and liver and between lung and testis.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura by extracting DNA from a sample obtained from a subject that had been exposed to the test agent 30 days or less prior to the sample being obtained from the subject as suggested by Manjanatha. The skilled artisan would have been motivated to expose a subject to a test agent for less than 30 days and then obtain a sample from the subject since Manjanatha teaches that after four weeks of exposure they could detect acquired mutations in the lung tissue which is the tumor target tissue (page 454). Further Manjanatha teaches that mice receiving the highest dosages had to stop after three weeks of exposure due to neurotoxicity (i.e., hind leg paralysis and sluggish movement) (page 453). Additionally the skilled artisan would have been motivated to expose the test subject to the test agent for 30 days or less because shorter exposure times reduce cost, efforts, and animal welfare. Further it would have been obvious to examine the mutation signature of a test agent that varies based on sample type for the benefit of being able to determine target organs for tumor induction (abstract). Finally it would have been obvious to use a transgenic animal since big blue mice, which are engineered to carry multiple copies of cll bacterial gene within their genome are commonly used in toxicology studies to detect mutations as demonstrated by Manjanatha (para 0016).
The combined references do not teach that the sequencing method is duplex sequencing (clm 13). The combined references do not teach a method wherein duplex sequencing DNA fragments includes duplex sequencing one or more targeted genomic regions (clm 72).
However Schmitt discloses a sequencing method called Duplex Consensus Sequencing (DCS). Schmitt teaches that this method greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand (abstract). Schmitt teaches methods that allow for targeted sequencing of a specific region of the genome (para 0062).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura and Manjanatha by sequencing using the Duplex Consensus Sequencing method of Schmitt. The skilled artisan would have been motivated to use DCS particularly since Schmitt teaches that this that this method greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand (abstract). Additionally it would have been obvious to use a method that allows for target sequencing of a specific region of the genome for the benefit being able to avoid sequencing the entire genome which is more costly and requires more time.
The combined references do not teach a method wherein the mutation signature is a triplet mutation signature (clm 13).
However Alexandrov discloses triplet mutation signatures. Alexandrov teaches they extracted mutational signatures using base substitutions and additionally included information on the sequence context of each mutation. Because there are six classes of base substitution—C>A, C>G, C>T, T>A, T>C, T>G (all substitutions are referred to by the pyrimidine of the mutated Watson– Crick base pair)—and as we incorporated information on the bases immediately 5’ and 3’ to each mutated base, there are 96 possible mutations in this classification. This 96 substitution classification is particularly useful for distinguishing mutational signatures that cause the same substitutions but in different sequence contexts. Alexandrov teaches applying this approach to the 30 cancer types revealed 21 distinct validated mutational signatures. These show substantial diversity (Fig. 2 and Supplementary Figs 2–23). There are signatures characterized by prominence of only one or two of the 96 possible substitution mutations, indicating remarkable specificity of mutation type and sequence context (signature 10). By contrast, others exhibit a more-or-less equal representation of all 96 mutations (signature 3). There are signatures characterized predominantly by C>T (signatures 1A/B, 6, 7, 11, 15, 19), C>A (4, 8, 18), T>C (5, 12, 16, 21) and T>G mutations (9, 17), with others showing distinctive combinations of mutation classes (2, 13, 14) (page 417, Fig 2, Fig 3, Supplemental Fig 2-23). Supplemental Figures 2-23 show 21 triplet mutation signatures. For example Signature 10 is show below:
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This signature has a high number of C>A mutations in the TCT context and a high number of C>T mutations in the TCG context.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Manjanatha, and Schmitt by determining a triplet mutation signature for the test agent as suggested by Alexandrov. The skilled artisan would have been motivated to determine a triplet mutation signature of a test agent since Alexandrov teaches that they are particularly useful for distinguishing mutational signatures that cause the same substitutions but in different sequence contexts.
7. Claims 66 is rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Manjanatha (EP 2706123 Pub 12/3/2014), Schmitt (US 2015/0044687 Pub 2/12/2015), and Alexandrov (Nature 500 415-421 2013) as applied to claim 13 above and in further view of Poon (Genome Medicine 2014 6:24 pages 1-14).
The teachings of Matsumura, Manjanatha, Schmitt, and Alexandrov are presented above.
The combined references do not teach comparing the mutation signature of the test agent with mutation signatures of one or more known genotoxins (clm 66).
However Poon teaches establishing connections between specific mutagens and their mutation signatures. Poon teaches that this requires experimental exposure of cells or animals to mutagens or their biochemically active metabolites, followed by next-generation sequencing of either clonal populations of exposed cells or of tumors that develop in exposed animals. Sequencing of the exposed genomes will connect specific mutagens to their mutation signatures in far more detail than is currently available. When mutation signatures cannot be found among the signatures of known mutagens, this would suggest the effects of an unknown exposure or mutational process, and point to the need for further epidemiological, toxicological, or biological research (page 11). Additionally Poon further teaches that mutation signatures can be correlated with strength to exposure of the test agent (Fig 1) and that mutation signatures may be different in different tissues and cell types (page 12).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Manjanatha, Schmitt, and Alexandrov by comparing the mutation signature of the test agent with mutation signatures of one or more known genotoxins as suggested by Poon. The skilled artisan would have been motivated to compare the mutation signature of a test agent with the mutation signature of known genotoxins for the benefit of being able to determine if the test agent is likely to cause mutagenesis.
8. Claim 70 is rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Manjanatha (EP 2706123 Pub 12/3/2014), Schmitt (US 2015/0044687 Pub 2/12/2015), and Alexandrov (Nature 500 415-421 2013) as applied to claim 13 above and in further view of Sintchenko (Nature June 2007 Vol 5 pages 464-470).
The teachings of Matsumura, Manjanatha, Schmitt, and Alexandrov are presented above.
The combined references do not teach a method wherein the mutagenic signature is generated by computational pattern matching (clm 70).
However Sintchenko discloses using computation pattern matching (page 467, middle column).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Manjanatha, Schmitt, and Alexandrov by using computation pattern matching to obtained the mutagenic signature as suggested by Sintchenko particularly since this was a known way to analyze nucleic acid sequence information.
9. Claims 73 and 74 are rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Manjanatha (EP 2706123 Pub 12/3/2014), Schmitt (US 2015/0044687 Pub 2/12/2015), and Alexandrov (Nature 500 415-421 2013) as applied to claims 13 and 72 above and in further view of Nguyen (Stem Cells International Vol 2016 Article ID 1346521).
The teachings of Matsumura, Manjanatha, Schmitt, and Alexandrov are presented above.
The combined references do not teach a method wherein the targeted genomic region is a mutation-prone site in the genome (clm 73). The combined references do not teach a method wherein the targeted genomic region comprises a known cancer driver gene (clm 74).
However Nguyen teaches that ionizing radiation (IR) is a known mutagen that is widely employed for medical diagnostic and therapeutic purposes. To study the extent of genetic variations in DNA caused by IR, we used IR-sensitive human embryonic stem cells (hESCs). Four hESC cell lines, H1, H7, H9, and H14, were subjected to IR at 0.2 or 1 Gy dose and then maintained in culture for four days before being harvested for DNA isolation. Since IR is often implicated as a risk for inducing cancer, a primer pool targeting genomic “hotspot” regions that are frequently mutated in human cancer genes was used to generate libraries from irradiated and control samples. Using a semiconductor-based next-generation sequencing approach, we were able to consistently sequence these samples with deep coverage for reliable data analysis (abstract).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Manjanatha, Schmitt, and Alexandrov by sequencing targeted genomic regions that are mutation prone and present in cancer driver genes as suggested by Nguyen. Based on the teachings of Nguyen the skilled artisan would have been motivated to target cancer “hotspot” regions of the genome for the benefit of being able to determine if the test agent is potentially carcinogenic.
10. Claim 76 is rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Manjanatha (Environmental and Molecular Mutagenesis 56:446-456 (2015), Schmitt (US 2015/0044687 Pub 2/12/2015), and Alexandrov (Nature 500 415-421 2013) as applied to claim 13 above and in further view of Meils (EP 2706123 Pub 3/12/2014).
The teachings of Matsumura, Manjanatha, Schmitt, and Alexandrov are presented above.
The combined references do not teach a method wherein the subject is a non-transgenic animal and wherein the DNA fragments comprise endogenous genomic regions.
However Melis teaches an in vitro method for determining whether it is likely or unlikely that a compound is a genotoxic carcinogen wherein the expression level of a marker gene is determined in a sample obtained from a rodent previously exposed to the compound (para 0013). Melis teaches that the method may be performed with any kind of rodent and discloses that C57BL/6J mice and C57BLC6 mice are suitable (para 0016).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Manjanatha, Schmitt, and Alexandrov by using a non-transgenic animal and detecting endogenous genomic regions as suggested by Meilis. One of skill in the art would have been motivated to perform laboratory testing on non-transgenic animals such as C57BL/6J mice and C57BLC6 because these non-transgenic animals are much cheaper to purchase in comparison to their transgenic counterparts.
11. Claims 25, 79, 82-85, 87-90, and 93-95 are rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Schmitt (US 2015/0044687 Pub 2/12/2015), Manjanatha (Environmental and Molecular Mutagenesis 56:446-456 (2015), Poon (Genome Medicine 2014 6:24 pages 1-14), and Alexandrov (Nature 500 415-421 2013).
Regarding Claim 25 Matsumura teaches a method for evaluating genotoxicity of a substance. The method comprises (1) obtaining DNAs from a test group, the test group is a cell population exposed to the test substance; (2) sequencing fragments of the DNAs to obtain one or more read sequences per fragment; (3) comparing each of the one or more read sequences with a reference sequence to detect sites of mismatch bases between the each read sequence and the reference sequence, wherein the reference sequence is a known sequence in the DNAs; (4) obtaining the sites detected in the step (3) as sites of mutation; (5) classifying each of the obtained mutations according to mutation patterns; and (6) determining respective mutation frequencies of the mutation patterns obtained in the step (5) (para 0017). Matsumura teaches obtaining information on a mutation at the mutation site. Examples of the information to be obtained include, but are not limited to, the type of the mismatched site (mutation site) (e.g., a substitution site, a deletion site or an insertion site), the type of the base at the site and the type of a base (or a base pair) before the mutation (e.g., the type of a base at a position corresponding to the site on the reference sequence), and the types of both adjacent bases of the site (e.g., the types of both bases adjacent to the position corresponding to the site on the reference sequence) (para 54). Matsumura further teaches that that the obtained pieces of information can be collected to create a database for mutation analysis. For example, a database of all information on the mutation site obtained from each read sequence may be created; a database may be created in which mutation information obtained from each read sequence is classified by type of the mutation site; a database may be created in which mutation information obtained from each read sequence is classified by type of a base before the mutation (e.g., in the reference sequence) or after the mutation at the mutation site; a database may be created in which mutation information obtained from each read sequence is classified by base length of the mutation site (e.g., length of an insertion, deletion or substitution site); or a database may be created by combining these classifications. Alternatively, a database may be created together with information on whether the identified position of the mutation site on the genome corresponds to a coding region or a noncoding region of a gene and, if the mutation site is in the coding region, information on whether the coding region is an intron or an exon or resides in a strand to be transcribed into RNA or not (para 0056). Thus Matsumura teaches a method comprising: sequencing DNA fragments from a biological source exposed to the test agent; and determining a mutation signature of the test agent including at least one of a mutation pattern, a mutation type, a mutant frequency, a mutation type distribution, and a genomic distribution of mutations in the sample.
Regarding 79 Matsumura teaches that examples of the cell population used in the method of the present invention include specimens harvested from animals or plants, and populations of animal-, plant- or microbe-derived cultured cells and preferably include populations of animal, plant or microbe strain-derived cultured cells (para 0105). Thus Matsumura teaches a method wherein the biological source is cells grown in culture.
Regarding Claim 83 Matsumura teaches adding the test substance to a medium containing a cell population (para 0104). Thus Matsumura teaches a method comprising exposing the biological source to the test agent.
Regarding Claim 84 Matsumura teaches a method for evaluating the genotoxicity of a test substance wherein a population of cancer cells is used as the test group (para 0095). Thus Matsumura teaches a method wherein prior to exposing the biological source to the test agent, the biological source is or comprises a cancer tissue.
Regarding Claim 85 Matsumura teaches a method for evaluating the genotoxicity of a test substance wherein a population of non-cancer cells is used as a control group (para 0095). Thus Matsumura teaches a method wherein prior to exposing the biological source to the test agent, the biological source is healthy tissue.
Regarding Claim 86 Matsumura teaches a method wherein the sample is blood (para 0098).
Regarding Claim 87 Matsumura teaches that the sample is a cancer cell line (para 0107).
Regarding Claim 88 Matsumura teaches a method wherein the biological source comprises cancerous cells and the test agent is tested for genotoxicity in the cancer cells (0095).
Regarding Claim 89 Matsumura teaches that the evaluation of medicines, cosmetics, various chemical substances, etc. for their genotoxicity is important for public safety (para 0003). Thus Matsumura teaches a method wherein the test agent is a therapeutic compound or a pharmaceutical composition or formulation.
Regarding Claim 90 Matsumura teaches determining one or more of a mutant frequency and a mutation signature for the portion of the cancerous cells prior to exposure to the therapeutic compound (para 0079).
Regarding Claim 93 Matsumura teaches that the evaluation of medicines, cosmetics, various chemical substances, etc. for their genotoxicity is important for public safety (para 0003). Thus Matsumura teaches a method wherein the test agent comprises a drug (medicine) or a cosmetic substance.
Regarding Claim 94 Matsumura teaches the test substance used in the method for evaluating the genotoxicity of a test substance according to the present invention is not particularly limited as long as the substance is to be evaluated for its genotoxicity. Matsumura further teaches that the test substance may be a compound or may be a composition or a mixture (para 0103). Thus Matsumura teaches a method wherein the test agent is an agent or factor with unknown genotoxicity and is selected from a compound.
Regarding Claim 95 Matsumura teaches that the evaluation of medicines, cosmetics, various chemical substances, etc. for their genotoxicity is important for public safety (para 0003). Thus Matsumura teaches a method wherein the test agent is a therapeutic compound or a pharmaceutical composition or formulation.
Matsumura does not teach (a) preparing a sequencing library from a sample comprising a plurality of double-stranded DNA fragments from a biological source exposed to the test agent, wherein preparing the sequence library comprises ligating asymmetric adapter molecules to the plurality of double-stranded DNA fragments to generate a plurality of adapter-DNA molecules; (b) sequencing first and second strands of the adapter-DNA molecules to provide a first strand sequence read and a second strand sequence read for at least a portion of the adapter-DNA molecules;(c) for each adapter-DNA molecule in the portion, comparing the first strand sequence read and the second strand sequence read to identify one or more correspondences between the first and second strand sequence reads; and (d) determining a mutation signature of the test agent (clm 25). Matsumura does not teach associating the first strand sequence read with the second strand sequence read using an adapter sequence.
However Schmitt discloses a sequencing method called Duplex Consensus Sequencing (DCS). Schmitt teaches that this method greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand (abstract). Schmitt, in FIG. 1 illustrates an overview of Duplex Consensus Sequencing. Sheared double-stranded DNA that has been end-repaired and T-tailed is combined with A-tailed SMI adaptors and ligated according to one embodiment. Because every adaptor contains a unique, double-stranded, complementary n-mer random tag on each end (n-mer=12 bp according to one embodiment), every DNA fragment becomes labeled with two distinct SMI sequences (arbitrarily designated .alpha. and .beta. in the single capture event shown). After size-selecting for appropriate length fragments, PCR amplification with primers containing Illumina flow-cell-compatible tails is carried out to generate families of PCR duplicates. By virtue of the asymmetric nature of adapted fragments, two types of PCR products are produced from each capture event. Those derived from one strand will have the α SMI sequence adjacent to flow-cell sequence 1 and the β SMI sequence adjacent to flow cell sequence 2. PCR products originating from the complementary strand are labeled reciprocally (para 0012). Schmitt teaches consensus sequences arising from two complementary strands of duplex DNA can be identified by virtue of the complementary SMIs (FIG. 3) to identify the "partner SMI." Specifically, a 24-nucleotide SMI consists of two 12-nucleotide sequences that can be designated XY. For an SMI of form XY in read 1, the partner SMI will be of form YX in read 2. An example to illustrate this point is given in FIG. 4. Following partnering of two strands by virtue of their complementary SMIs, the sequences of the strands are compared. Sequence reads at a given position are kept only if the read data from each of the two paired strands is in agreement (para 0129).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura by sequencing using the Duplex Consensus Sequencing method of Schmitt. The skilled artisan would have been motivated to use DCS particularly since Schmitt teaches that this that this method greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand (abstract).
The combined references do not teach a method wherein the biological source was exposed to the test agent 30 day or less prior to extracting the sample comprising a plurality of double stranded DNA fragments (clm 25).
However Manjanatha teaches that to investigate whether or not gene mutation is involved in the etiology of acrylamide (AA) or glycidamide (GA) induced mouse lung carcinogenicity, we screened for cll mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19–111 mg/kg bw/days). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P 0.01). The predominant types of mutations in the lung cll gene from AA- and GA treated mice were A:T → T:A, and G:C →C:G transversions, and -1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted (abstract).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura and Schmitt by exposing the biological source to the test agent 30 days or less prior to extracting the sample as suggested by Manjanatha. The skilled artisan would have been motivated to expose a biological source to a test agent for less than 30 days and then obtain a sample from the source since Manjanatha teaches that after four weeks of exposure they could detect acquired mutations in the lung tissue which is the tumor target tissue (page 454). Further Manjanatha teaches that mice receiving the highest dosages had to stop after three weeks of exposure due to neurotoxicity (i.e., hind leg paralysis and sluggish movement) (page 453). Additionally the skilled artisan would have been motivated to expose the test subject to the test agent for 30 days or less because shorter exposure times reduce cost, efforts, and animal welfare.
The combined references do not teach comparing the mutation signature of the test agent to a plurality of mutation signatures derived from known genotoxins to determine if the mutation signature is sufficiently similar to a mutation signature from a known genotoxin (clm 25).
However Poon teaches establishing connections between specific mutagens and their mutation signatures. Poon teaches that this requires experimental exposure of cells or animals to mutagens or their biochemically active metabolites, followed by next-generation sequencing of either clonal populations of exposed cells or of tumors that develop in exposed animals. Sequencing of the exposed genomes will connect specific mutagens to their mutation signatures in far more detail than is currently available. When mutation signatures cannot be found among the signatures of known mutagens, this would suggest the effects of an unknown exposure or mutational process, and point to the need for further epidemiological, toxicological, or biological research (page 11).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Schmitt, and Manjanatha by comparing the mutation signature of the test agent with mutation signatures of one or more known genotoxins as suggested by Poon. The skilled artisan would have been motivated to compare the mutation signature of a test agent with the mutation signature of known genotoxins for the benefit of being able to determine if the test agent is likely to cause mutagenesis and particularly mutagenesis that results in cancer.
The combined references do not teach a method wherein the mutation signature is a triplet mutation signature (clm 25).
However Alexandrov discloses triplet mutation signatures. Alexandrov teaches they extracted mutational signatures using base substitutions and additionally included information on the sequence context of each mutation. Because there are six classes of base substitution—C>A, C>G, C>T, T>A, T>C, T>G (all substitutions are referred to by the pyrimidine of the mutated Watson– Crick base pair)—and as we incorporated information on the bases immediately 5’ and 3’ to each mutated base, there are 96 possible mutations in this classification. This 96 substitution classification is particularly useful for distinguishing mutational signatures that cause the same substitutions but in different sequence contexts. Alexandrov teaches applying this approach to the 30 cancer types revealed 21 distinct validated mutational signatures. These show substantial diversity (Fig. 2 and Supplementary Figs 2–23). There are signatures characterized by prominence of only one or two of the 96 possible substitution mutations, indicating remarkable specificity of mutation type and sequence context (signature 10). By contrast, others exhibit a more-or-less equal representation of all 96 mutations (signature 3). There are signatures characterized predominantly by C>T (signatures 1A/B, 6, 7, 11, 15, 19), C>A (4, 8, 18), T>C (5, 12, 16, 21) and T>G mutations (9, 17), with others showing distinctive combinations of mutation classes (2, 13, 14) (page 417, Fig 2, Fig 3, Supplemental Fig 2-23). Supplemental Figures 2-23 show 21 triplet mutation signatures. For example Signature 10 is show below:
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This signature has a high number of C>A mutations in the TCT context and a high number of C>T mutations in the TCG context.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Schmitt, Manjanatha, and Poon by determining a triplet mutation signature for the test agent as suggested by Alexandrov. The skilled artisan would have been motivated to determine a triplet mutation signature of a test agent since Alexandrov teaches that they are particularly useful for distinguishing mutational signatures that cause the same substitutions but in different sequence contexts.
12. Claim 77 is rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Schmitt (US 2015/0044687 Pub 2/12/2015), Manjanatha (Environmental and Molecular Mutagenesis 56:446-456 (2015), Poon (Genome Medicine 2014 6:24 pages 1-14), and Alexandrov (Nature 500 415-421 2013) as applied to claim 25 above and in further view of Jenkins (Mutagenesis vol 20 no 6 pages 389-398 2005).
The teachings of Matsumura, Schmitt, Manjanatha, Poon, and Alexandrov are presented above.
The combined references do not teach determining if the mutation signature of the test agent comprises mutant frequency above a safe threshold frequency (clm 77).
However Jenkins teaches the concept of using dose response thresholds for genotoxic alkylating agents (abstract).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Schmitt, Manjanatha, Poon, and Alexandrov by determining if a mutation signature of a test agent has a mutation frequency above or below a safe threshold frequency as suggested by Jenkins. Jenkins teaches that the demonstration and acceptance of dose response thresholds for genotoxins may have substantial implications for the setting of safe exposure levels (abstract). Based on these teachings it would have been obvious to compare the mutation frequency of a test agent to a present safety threshold for the benefit of being able to determine the amount of test agent exposure that is safe or not safe.
13. Claims 91-92 are rejected under 35 U.S.C. 103 as being unpatentable over Matsumura (US 2019/0259469 claiming priority to PCT/JP2017/005700 Filed on 2/16/2017) in view of Schmitt (US 2015/0044687 Pub 2/12/2015), Manjanatha (Environmental and MMolecularMutagensis 56:446-456 (2015), Poon (Genome Medicine 2014 6:24 pages 1-14), and Alexandrov (Nature 500 415-421 2013) as applied to claim 25 and above and in further view of Sasaki (Mutation Research 1997 Vol 391 pages 201-214)
The teachings of Matsumura, Schmitt, Manjanatha, Poon, and Alexandrov are presented above.
The combined references do not teach a method wherein the test agent is assessed for genotoxicity to at least a portion of the biological source (clm 91). The combined references do not teach a method comprising determining if the portion of the biological source is sensitive to the genotoxicity of the test agent, and wherein the method further comprises determining a mutation signature for the portion of the biological source (clm 92).
However Sasaki teaches that they assessed the genotoxicity of 8 rodent hepatic carcinogens in 5 mouse organs liver, lung, kidney, spleen, and bone marrow . The carcinogens we studied were p-aminoazobenzene, auramine, 2,4-diaminotoluene, p-dichlorobenzene, ethylene thiourea ETU , styrene-7,8-oxide, phenobarbital sodium, and benzene-1,2,3,4,5,6-hexachloride BHC ; except for p-aminoazobenzene, they do not induce micronuclei in mouse bone marrow cells. Mice were sacrificed 3 and 24 h after the administration of each carcinogen. p-Aminoazobenzene, ETU, and styrene-7,8-oxide induced alkaline labile DNA lesions in all of the organs studied. Auramine, 2,4-diaminotoluene, p-dichlorobenzene, and phenobarbital sodium also produced lesions, but their effect was greatest in the liver. BHC, which is not genotoxic in in vitro tests, did not show any effects.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Matsumura, Schmitt, Manjanatha, Poon, and Alexandrov by assessing for genotoxicity to multiple organs and making a mutation signature for each organ. Based on the teachings of Saski the skilled artisan would have recognized that test agents have different mutagenic effects on different organs and the skilled artisan would have been motivated to look at different organs prior to declaring any specific test agent genotoxic or non-genotoxic.
Response To Arguments
14. In the response the Applicants traversed the rejection of independent claim 13 under 35 USC 103 over the prior art combination of Matsumura, Melis, Schmitt, and Alexandrov. The Applicants note that previously claim 69 recited “wherein the subject was exposed to the test agent 30 days or less prior to the sample being obtained from the subject”. The Applicants note that claim 69 has been canceled and this limitation has been moved into independent claim 13.
Additionally the Applicants traversed the rejection of independent claim 25 under 35 USC 103 over the prior art combination of Matsumura, Schmitt, Poon and Alexandrov. The Applicants note that previously claim 80 recited “wherein the subject was exposed to the test agent 30 days or less prior to the sample being obtained from the subject”. The Applicants note that claim 80 has been canceled and this limitation has been moved into independent claim 25.
The Applicants state that Melis teaches an in vitro method for determining whether it is likely or unlikely that a compound is a genotoxic carcinogen wherein the expression level of a marker gene is determined in a sample obtained from a rodent previously exposed to the compound," including after time periods "including 2, 3, or 4 weeks. They argue that Melis discloses measuring expression levels of marker genes to distinguish genotoxic or carcinogenic compounds, rather than mutation signatures as claimed. They argue that gene expression measurements capture a completely different set of information than assessments of mutation signatures. They argue that expression level changes of marker genes generally occur and quantifiably amplify much faster than mutations can be detected and therefore, lend to a much earlier timeframe for measurement, e.g., 30 days or less after exposure. They argue that genomic mutations require a much higher mutation burden to potentially detect. They argue that the Examiner fails to explain why a skilled artisan would refer to the exposure time period disclosures of Melis, which are again directed to gene expression measurement, for methods of generating a mutational signature wherein the subject or biological source was exposed to the test agent 30 days or less.
This argument has been fully considered. The amendment of claim 13 to recite “wherein the subject was exposed to the test agent 30 days or less prior to the sample being obtained from the subject” changes the scope of claims 13, 66-68, 70, and 72-76. The amendment of claim 25 to recite “wherein the biological source was exposed to the test agent 30 days or less prior to the sample being obtained from the subject” changes the scope of claims 25, 77, 79, 82-95. All of the previously presented rejections have been modified to address the claims as amended. The rejections now rely on the prior art of Manjanatha (Environmental and Molecular Mutagenesis 56:446-456 (2015) to teach that the subject/biological source was exposed to the test agent 30 days or less prior to the sample being obtained from the subject.
15. 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 extension fee 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 date of this final action.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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