DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 6/25/2025 has been entered.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claim 171 has been added. Claim 151 has been canceled. Claims 152-170 remain withdrawn. Claims 1-15, 33-39, and 171 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 4/4/2025, 6/27/2025, and 9/17/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Response to Applicant’s Amendments
Claim Objections
Claim 1 was objected to for various informalities. In light of Applicant’s amendments to the claims submitted 6/25/2025, this objection has been withdrawn.
35 USC 101 Rejections
Claims 1-15, 33-39, and 151 were rejected for reciting judicial exceptions without significantly more. After further search and consideration, these rejections have been withdrawn for all currently pending claims. Claim 151 has been canceled, so this rejection has been rendered moot. See new grounds of rejection below. See also “Response to Applicant’s Arguments” below.
35 USC 103 Rejections
Claims 1-15, 33-39, and 151 were rejected over Kincaid et al. (WO 2019/140201), as evidenced by Thermo Fisher Scientific (DNase I Product Information, 2016), in view of Maurano et al. (Science, 2012) and various combinations of references. After further search and consideration, these rejections have been withdrawn for all currently pending claims. Claim 151 has been canceled, so this rejection has been rendered moot. See new grounds of rejection below.
Response to Applicant’s Arguments
Election/Restriction
Applicant states that in the Office Action mailed 6/12/2024, claims 152-170 were withdrawn with no reasoning. This Office Action was a Non-Final Rejection that noted that Applicant elected a group of claims (claims 1-15 and 33-39) without traverse in their response to the Restriction Requirement mailed 2/2/2024. The initial restriction requirement was done under the guidance provided by 35 USC 121 and is considered proper. Applicant added claims 152-170 in the claims submitted along with their response to this initial restriction requirement. Claims 152-170 and the currently elected claims are related as product and process of use. The inventions are distinct because the process need not be performed with the specific product, as the process can be done at least partially in the human mind (as noted by the “Response to Applicant’s Arguments” and the 35 USC 101 Rejections below) and without the use of an assay device (e.g. analyzing the biological sample in a sample tube or tubes not attached to an assay device). Thus, the withdrawal of these claims is also considered proper.
35 USC 101 Rejections
Regarding the 35 USC 101 Rejections, Applicant argues that the claims integrate the listed judicial exceptions into a practical application that is an improvement to a technical field, specifically in providing a determination of the level of abnormality in a sample. Applicant argues that the claimed invention does in fact demonstrate said improvement, particularly in that a “first parameter” based on cutting signatures for a nuclease provides improved cancer detection accuracy over using a motif diversity score (Remarks, pages 14-15). Applicant also states that Yin does not adequately detail that the claimed method is conventional (Remarks, pages 15-16).
As noted in the previous 35 USC 101 Rejections, claim 1 contains multiple identifying and determining steps. In particular, the final three determining steps may still be mental processes that could be performed in the human mind or with the use of a physical aid, such as pen and paper. There is no minimum or maximum amount for how many sequence reads must be included in the “first amount,” and so this step could amount to counting a small number of sequence reads. Determining a “first parameter” based on this “first amount” could amount to a simple mathematical calculation, such as a sum or mean, and determining a classification could be as simple as comparing the first parameter to other numbers. Thus, these steps are recited at a high level of generality without any specific techniques that would render them unable to practically be done in the human mind. See MPEP 2106.04(a)(2) III B.
As to Applicant’s assertion that the instant invention is an improvement to technology, Applicant references paras. 3, 177, and 183 of the instant specification. Para. 3 discusses previous techniques for analyzing nuclease expression, para. 177 describes very generally that embodiments of the invention may be less invasive and more accurate than other methods, and para. 183 compares accuracy between one embodiment of the present invention and one other type of method. Applicant states that these teachings are commensurate in scope with claim 1, particularly with regard to para. 183 of the instant specification. Para. 183 discusses specific nucleases (DNASEIL3 and DFFB), in association with a particular disease (HCC) and uses a specific parameter (a cutting signature ratio). These nucleases, disease, and parameter are not specifically disclosed in claim 1, and so any potential improvement described by this paragraph in the instant specification is not commensurate in scope with the invention of claim 1, and so does not persuasively suggest the claimed method is an improvement upon the technology of the prior art. See MPEP 2103.05(a) II.
As to Applicant’s arguments against the use of Yin, as a result of further search and consideration of claim 1 due to the claim amendments, additional prior art was found that is now incorporated into the 35 USC 101 rejections, and so these arguments have been rendered moot.
35 USC 103 Rejections
Regarding the prior art rejections, Applicant argues that Kincaid does not use blunt ended sequences for cancer classification, and thus does not identify a first set of the at least 10,000 sequence reads including a particular end signature where at least a portion of the reads are blunt ended, as stated in claim 1. Applicant also states that Kincaid does not teach that end signatures can be used for abnormality classification, and thus, does not use an amount of sequence reads to determine a parameter and thus determine an abnormality classification as claimed. Regarding Maurano, Applicant argues that the reference does not teach that DNase I is differentially regulated in abnormal cells as claimed, and that Kincaid does not teach the new limitations presented in instant claim 171 (Remarks, pages 17-20).
The Examiner has provided new grounds of rejection below that do not use Kincaid as a primary reference, and moreover do not use Kincaid in the rejection of claim 1. These new rejections also do not use Maurano to teach expression activity of a nuclease. Therefore, these arguments have been rendered moot.
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 1-15, 33-39, and 171 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception without significantly more. The claims recite a natural law and abstract ideas.
Claims 1 and 3 are directed to a method of classifying a level of abnormality in a subject involving the use of a nuclease that preferentially cuts DNA (or two nucleases, in the case of claim 3). The abstract ideas recited are the determining steps of the method, as these encompass mental processes, as stated above in the “Response to Applicant’s Arguments” section. As written, these steps are recited at a high level of generality without any specific techniques that would render them unable to practically be done in the human mind or by a human with a physical aid. See MPEP 2106.04(a). The law of nature recited is the relationship between the expression of the nuclease and the presence of an abnormality in the one or more tissue types. These judicial exceptions are not integrated into a practical application because there is not a required treatment step or anything else that would integrate the method into a practical application. See MPEP 2106.04(d)(2). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not require any additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Jiang et al. (Cancer Discovery, 2020). Jiang teaches analyzing plasma end motifs in patients with hepatocellular carcinoma (HCC; Abstract), specifically examining DNASE1L3 (page 665, column 2, para. 1). Such a method involves analyzing plasma DNA fragments treated with a nuclease, where said treatment may produce segments with both overhangs and blunt ends (see Figure 1 and Figure 1 caption). DNASE1L3 expression was found to differ between HCC tumor tissues and normal tissues, with expression being downregulated in cancer tissues (Figures 2A). Examining plasma end motifs in control vs. HCC individuals, the end motif CCCA was found to occur less often in HCC individuals (Figure 2B). The CCCA motif was the most common in healthy controls (page 666, columns 1-2 joining para.). Plasma DNA was analyzed, which contained cfDNA and provided information about their tissue of origin (page 665, column 1, paras. 1-2 and page 669, column 2, para. 2). Sequencing analysis was performed, where all end motifs were analyzed from sequence reads, and around 2% of HCC reads were found to have the CCCA end motif. HCC patients were also found to have significantly greater motif diversity scores compared to control groups (Figure 3 and page 667, column 1, para. 3). Jiang then used motif diversity scores across DNA samples for other cancer types and also found significantly higher values compared to control groups (page 668, column 1, para. 5). The reference then attempted to use motif diversity scores for cancer detection, where the scores were used to generate AUC values. This classifier proved to be a good predictor provided that enough DNA molecules were sequenced and enough tumor DNA fraction was presented in the samples (Figure 3G-H and page 669, “Classification Performance Using Plasma DNA End Motifs”). In mapping the reference to the claim, Jiang teaches mechanisms for sequencing many more than 10,000 molecules (and teaches sequencing up to 30,000,000). This reference renders the method steps of claims 1 and 3 conventional (see also the 35 USC 103 Rejections below). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, claims 1 and 3 are directed to judicial exceptions without significantly more.
Claim 2 depends on claim 1 and further specifies how the determination of the level of abnormality in the sample is made. This limitation is an extension of the abstract ideas recited in claim 1, and does not provide any specific techniques that would render the determination unable to practically be done in the human mind. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claim 4 depends on claim 3 and discusses how the nucleases used may be differentially expressed in abnormal cells (i.e. upregulated or downregulated). This claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Jiang as described above, as this claim is also mainly drawn to evaluating nuclease activity. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 3.
Claim 5 depends on claim 1 and is similar to claim 3 in that a second nuclease is required. The main difference between claims 5 and 3 is in how the count of the second sequence reads is used. In claim 5, it is used to aid in determining the first parameter described in claim 1. This limitation is still an extension of the abstract idea recited in claim 1, and does not provide any specific techniques that would render the determination unable to practically be done in the human mind. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claim 6 depends on claim 5 and discusses how the nucleases used may be differentially expressed in abnormal cells (i.e. upregulated or downregulated), similar to the manner recited in claim 4. This claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Jiang as described above, as this claim is also mainly drawn to evaluating nuclease activity. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 5.
Claim 7 depends on claim 1 and requires that fetal tissue be examined. This claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Zhang et al. (Journal of Histochemistry & Cytochemistry, 2008). This reference teaches taking samples of fetal tissue, placing them on microarrays, and performing staining (“Materials and Methods”). This involved standard procedures and commercially available kits and reagents. This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claims 8 and 9 depend on claim 1 and require that the subject be a pregnant female, placental tissue in maternal plasma be examined, and that the abnormality examined be preeclampsia, preterm birth, fetal chromosomal aneuploidies, or fetal genetic disorders. These claims do not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Lo et al. (Nature Medicine, 2007). This reference teaches measuring placental mRNA in maternal plasma in order to diagnose fetal aneuploidy (trisomy 21; Abstract). This involved methods of processing, assaying, and amplifying that included commercially available reagents, kits, microarrays, software, equipment, and primers and probes (see Lo Supplementary Methods). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, these claims are also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claim 10 depends on claim 1 and requires additional analyzing and determining steps related to a second biological subject. These limitations are similar to the abstract ideas recited in claim 1, and do not provide any specific techniques that would render the determination unable to practically be done in the human mind. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claims 11-13 depend on claim 1 and require that the abnormality examined be a pathology, specifically cancer in the case of claim 12, and specifically a pathology with multiple stages in the case of claim 13. These claims thus also only further define the judicial exceptions of claim 1 in that the abnormality analyzed in the method is further specified. Additionally, these claims do not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Elston et al. (Histopathology, 1991). This reference teaches methods of tissue preparation and tumor grading using histology, cell nuclei, and mitotic and tubule structures (pages 404-405, “Materials and methods”). Staining techniques were routine (page 404, “Tissue Preparation”), and analysis of samples was conducted with a commercially available microscope (page 405, “Mitotic counts”). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claims 14-15 depend on claim 11 and require that the abnormality be an auto-immune disorder, specifically systemic lupus erythematosus (SLE) in the case of claim 15. These claims thus also only further define the judicial exceptions of claims 1 and 11 in that the abnormality analyzed in the method is further specified. Additionally, these claims do not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Hanly et al. (Journal of Immunological Methods, 2010). This reference teaches using assays to detect autoantibodies to diagnose and assess patients with SLE (Abstract). These measurements occurred with commercially available screening tools, kits, and equipment, as well as standard procedures (page 76, “2.4 Measurement of autoantibodies”). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 11.
Claims 33 and 37 provide a list of nucleases that may be used in claims 1 and 3 respectively. These claims thus also only further define the judicial exceptions of claims 1 and 3 in that the differentially regulated nuclease(s) in abnormal cells that is/are analyzed in the method is further specified. Additionally, these claims do not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Jiang as described above, as these claims are also mainly drawn to evaluating nuclease activity. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claims 1 and 3.
Claim 34 depends on claim 33 and further specifies that the nuclease DNASEIL3 must be used, and that this nuclease must produce a particular end signature. This claim thus also only further defines the judicial exception of claim 33 in that the differentially regulated nuclease in abnormal cells that is analyzed in the method is further specified. Additionally, this claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Serpas et al. (PNAS, 2019). Serpas teaches DNASE1L3, and notes that this nuclease preferentially creates particular end motifs in nucleic acid fragments, including the end motif CCCA (Abstract and Table 1). This was examined in a previously described mouse model, and the nucleic acid analysis involved the use of commercially available kits, reagents, and equipment (page 648, column 1). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 33.
Claim 35 depends on claim 33 and further specifies that the nuclease DFFB must be used, and that this nuclease must produce a particular end signature. This claim thus also only further defines the judicial exception of claim 33 in that the differentially regulated nuclease in abnormal cells that is analyzed in the method is further specified. Additionally, this claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Widlak et al. (The Journal of Biological Chemistry, 2000), as evidenced by PhosphoSitePlus (2016). Widlak teaches the cleavage preferences of DFF40 (an alternative name for DFFB, see PhosphoSitePlus; Title). Widlak found that after cleavage with DFFB, the frequencies for the end signature were as follows: R (72%), R (74%), R (66%), Y (61%; page 8228, column 1, para. 1). This encompasses the sequence AAAT, which is one of the end signatures specified in claim 35. The methods of this study involved using previously recorded procedures and commercially available reagents (page 8227, “Assay for Endonuclease Activity” and “Analysis of Cleavage Sites”). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 33.
Claim 36 depends on claim 33 and further specifies that the nuclease DNase I must be used, and that this nuclease must produce a particular end signature. This claim thus also only further defines the judicial exception of claim 33 in that the differentially regulated nuclease in abnormal cells that is analyzed in the method is further specified. Additionally, this claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Drew et al. (Cell, 1984). Drew teaches methods related to the cleavage patterns of DNase I, DNase II, and copper-phenanthroline (Abstract). This reference teaches that DNase I preferentially cleaves sites, and can leave a TAAT end sequence (see Figure 1b, which shows increased activity for DNase I around nucleotides 12-15, which are shown in Figure 1a to be a TAAT, as well as Figure 2a-b, which both show the probability that DNase I can cleave after the TAAT sequence). These methods involved commercially available reagents with known equipment (pages 500-501, “Experimental Procedures”). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 33.
Claim 38 depends on claim 1 and further specifics that the analyzing step must be conducted with sequencing. This claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of Serpas, as this reference teaches sequencing DNA libraries using a commercially available platform and software (page 648, “DNA Sequencing Using the Illumina Platform”). This reference does not discuss any modifications to the commercial and standard aspects of these methods, providing evidence to the routineness of these methodologies. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claim 39 depends on claim 1 and further specifies the first parameter used. This limitation is still an extension of the abstract idea recited in claim 1, and does not provide any specific techniques that would render the determination unable to practically be done in the human mind. Thus, this claim is also directed to a judicial exception without significantly more for the reasons set forth above with respect to claim 1.
Claim 171 depends on claim 1 and requires that the first sequence end signature comprise more than one base. This claim does not further specify a treatment or other integrating step or involve additional elements that are not well-understood, routine, or conventional to those of ordinary skill in the art in view of at least Jiang, which teaches end signatures with four nucleotides, as described above.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 11-12, 33-34, 38-39, and 171 are rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al. (Cancer Discovery, 2020).
Jiang teaches analyzing plasma end motifs in patients with hepatocellular carcinoma (HCC; Abstract), specifically examining DNASE1L3 (page 665, column 2, para. 1). Such a method involves analyzing plasma DNA fragments treated with a nuclease, where said treatment may produce segments with both overhangs and blunt ends (see Figure 1 and Figure 1 caption). DNASE1L3 expression was found to differ between HCC tumor tissues and normal tissues, with expression being downregulated in cancer tissues (Figures 2A; instant claim 33). Examining plasma end motifs in control vs. HCC individuals, the end motif CCCA was found to occur less often in HCC individuals (Figure 2B). The CCCA motif was the most common in healthy controls (page 666, columns 1-2 joining para.; instant claims 34 and 171). Plasma DNA was analyzed, which contained cfDNA and provided information about their tissue of origin (page 665, column 1, paras. 1-2 and page 669, column 2, para. 2). Sequencing analysis was performed, and all end motifs were analyzed from sequence reads, and around 2% of HCC reads were found to have the CCCA end motif. HCC patients were also found to have significantly greater motif diversity scores compared to control groups (Figure 3 and page 667, column 1, para. 3; instant claim 38). Jiang then used motif diversity scores across DNA samples for other cancer types and also found significantly higher values compared to control groups (page 668, column 1, para. 5). The reference then attempted to use motif diversity scores for cancer detection, where the scores were used to generate AUC values. This classifier proved to be a good predictor provided that enough DNA molecules were sequenced and enough tumor DNA fraction was presented in the samples (Figure 3G-H and page 669, “Classification Performance Using Plasma DNA End Motifs”).
Overall, combining the various teachings presented in Jiang would render prima facie obvious the method of instant claim 1. Specifically, Jiang teaches the downregulated expression and preferential cutting of DNAS1L3 in patients with HCC, and then analyzes plasma DNA to obtain patterns related to cfDNAs involving sequencing. In mapping the reference to the claim, as Jiang teaches mechanisms for sequencing many more than 10,000 molecules (and teaches sequencing up to 30,000,000), and teaches determining a frequency of sequence reads that contain a particular motif, this would be equivalent to the “first set” of claim 1. The entirety of this “first set” (which would also be equivalent to the “first amount” of claim 1, as the “first amount” may be the entirety of the “first set”) was then used with other end motif frequences to create the motif diversity score (analogous to the “first parameter” of claim 1). This motif diversity score can then be used with sequencing data to provide an AUC value that can characterize a sample as being a classifier for HCC (equivalent to the “level of abnormality” in claim 1). Jiang does not specifically teach that the methods involving the sequencing of 30,000,000 fragments and AUC values is done with the same samples as the initial end motif and motif diversity score analyses. However, as motif diversity scores are used in the analysis shown in Figure 3G (see page 669, “Classification Performance Using Plasma DNA End Motifs”), it would be obvious to combine these methods to arrive at the invention of in the instant claims. Given that 30,000,000 is the sequencing limit provided by Jiang, and that 2% of sequences showed the preferential end motif of DNASEIL3, this would result in far more than 10,000 cfDNA sequences with an end motif of CCCA. By analyzing single plasma samples for end motifs, motif diversity scores, and AUC values as is done in Jiang, this would provide a minimally invasive way to diagnose a patient with HCC with a high degree of certainty. This could allow for earlier diagnosis, which would improve patient outcomes. These methods could also be used to measure motif diversity scores over time, which may indicate cancer progression and response to treatment, thereby also providing a minimally invasive way to gauge treatment efficacy, which can also improve patient outcomes. There would be a reasonable expectation of success in combining these teachings because they are all taught by Jiang, do not involve any fundamentally novel sample manipulation techniques (as methods of sample preparation and sequencing are well-known in the art), and simply involve combining various downstream computational analyses (instant claims 1 and 11-12).
Therefore, claims 1, 11-12, 33-34, 38, and 171 are prima facie obvious over Jiang.
Regarding claim 2, while the analysis in Figure 3G does not appear to utilize motif diversity score information from controls, Jiang teaches comparisons utilizing motif diversity scores from both HCC and non-cancer patients (Figure 3A and C) and does the same for analyses regarding liver transplantation and fetal-maternal DNA comparisons (see Figures 4A-C and E). Thus, it would also be prima facie obvious to take this comparison data into account in the method of Jiang described above in the rejection of claim 1, as these comparisons can provide additional data for making a determination of HCC in a patient. For example, if an AUC value is only marginally predictive (e.g. 0.75), also utilizing motif diversity score comparisons with known, non-cancerous samples could provide additional information to make diagnoses. The data from non-cancerous samples shown in Jiang would remain as a reference, and would not require additional sampling, and the motif diversity score for a patient would already be acquired in the initial patient sampling and analysis methods described above in the rejection of claim 1. This would then amount to simply comparing known values to one another, which would be possible for the ordinary artisan.
Therefore, claim 2 is prima facie obvious over Jiang.
Regarding claim 39, the motif diversity scores (the “first parameter” used in the rejection of claim 1) were created using the individual motif frequencies, where each frequency is put into a summation equation (see page 672, “MDS Calculation”). However, to obtain each motif frequency, the number of sequence reads containing said motif must be divided by the total number of sequence reads to obtain a percentage (see Figures 2B, 3B, and F). In the rejection of claim 1 over Jiang as described above, the “first amount” is just the number of sequence reads that contain the CCCA motif, and so creating these motif frequencies would involve a ratio of the first amount and the total amount of sequence reads. These motif frequencies can also be used to distinguish between HCC and non-cancerous samples (see Figures 2B, 3B, and F). Thus, to avoid additional calculations, the ordinary artisan would recognize that the motif frequencies for the end motifs that are significantly different between HCC and non-cancerous samples could also be used as a first parameter to detect a level of abnormality in a sample. This would be a less labor intensive analysis that may provide results faster, and could also be used in conjunction with motif diversity scores to provide a check on results/conclusions and enhance overall accuracy. If trends are different between motif frequencies and motif diversity scores, it may indicate a need for closer investigation or resampling. There would be a reasonable expectation of success in more closely examining the motif frequencies as these are already calculated in the method of Jiang, and so may simply require performing additional statistics that would be known to the ordinary artisan.
Thus, claim 39 is prima facie obvious over Jiang.
Claims 3-6 and 36-37 are rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al. (Cancer Discovery, 2020), and in view of Drew et al. (Cell, 1984) and Golonka et al. (JNCI Cancer Spectrum, 2019).
While Jiang does teach the methods of claims 1-2, 11-12, 33-34, 38-39, and 171 as described above, Jiang does not greatly discuss DNASE1. The reference does mention that this nuclease may also exhibit plasma DNA end preferences, but does not examine what these end preferences might be (page 671, column 2, para. 1).
Drew teaches methods related to the cleavage patterns of DNase I, DNase II, and copper-phenanthroline (Abstract). This reference teaches that DNase I preferentially cleaves sites, and can leave a TAAT end sequence (see Figure 1b, which shows increased activity for DNase I around nucleotides 12-15, which are shown in Figure 1a to be a TAAT, as well as Figure 2a-b, which both show the probability that DNase I can cleave after the TAAT sequence).
Golonka teaches that an increase in DNase I activity is associated with liver cancer (Abstract, page 5, column 1, para. 3, and Table 3). As noted above, Jiang teaches that DNASE1L3 is downregulated in hepatocellular carcinoma (Figure 1b and Table 1).
Regarding claim 36, prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Drew and Golonka to substitute the DNASIL3 used in the method of Jiang with DNASEI used by Drew and taught by Golonka. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” DNASEI is known, as well as its preferential end signatures and its differential expression in patients with liver cancer, and so its use in the method of Jiang would be very similar to that of DNASEIL3 – the only major change would be the end signature examined (TAAT). As these differences would be known before the method would be performed, the ordinary artisan would have a reasonable expectation of success in this substitution, and the results would be predictable.
Regarding claims 3-6 and 37, using the teachings of Drew and Golonka, it would be prima facie obvious to the ordinary artisan that two nucleases (DNASE1L3 and DNASE1) could be analyzed with the methods of Jiang described above. For the purposes of the claimed invention, there is no true distinction between the “first nuclease” and the “second nuclease,” and so this would simply equate to the use of one upregulated nuclease (DNASE1) and one downregulated nuclease (DNASEIL3) in liver cancer patients. Generally, this would provide additional information for diagnostics, which could lead to more accurate results, potentially saving time, stress, and resources for both practitioners and patients. As Drew and Golonka both demonstrate that analyzing the activity and end motif patterns related to DNASE1 are possible, there would be a reasonable expectation of success in additionally utilizing this nuclease in the method of Jiang.
Specifically regarding claims 3-4, it would therefore have been prima facie obvious for one of ordinary skill in the art to combine the teachings of Jiang with those of Drew and Golonka to perform the method of Jiang to produce and analyze sequence reads to provide end motif data on DNASEIL3 and DNASE1 to aid in determining the level of abnormality in sample tissues. These end motif data can be based on the known preferential end motifs of both of the nucleases (i.e. CCCA and TAAT), and by finding the frequencies of each of these motifs, these can be used as the parameters to determine if a subject has liver cancer, similar to the data shown in Figures 2B and 3B of Jiang. This would be useful because of the known information about the upregulation of DNASEI and downregulation of DNASEEIL3 in liver cancer – one would expect the frequency of the TAAT motif to go up compared to that of a wild-type sample and the frequency of the CCCA motif to go down compared to that of a wild-type sample. These motif frequencies can thus be used as separate parameters to gauge the likelihood that a particular patient has liver cancer. By analyzing data in relation to both nucleases, this can provide greater confidence to any conclusions drawn, and can also reveal if additional testing needs to be performed. For example, if the trends related to only one end motif are as expected, it may indicate a need for additional or replicate testing. This combination of teachings is simply using the guidance provided by Drew and Golonka with the method of Jiang to examine a second end motif more closely, and as Jiang does analyze each possible end motif, there would thus be a reasonable expectation of success.
Specifically regarding claims 5-6, as noted above, it would be prima facie obvious to examine both DNASEIL3 and DNASE1 in liver cancer patients. In the rejection of claim 1 above, the “first parameter” used is the motif diversity score. This motif diversity score utilizes each of the motif frequencies in a summation equation (page 672, “MDS Calculation”). Thus, the first parameter utilizes the sequence reads from both the preferential end motifs of DNASEIL3 and DNASE1, and the amount of the reads with these particular end motifs would be analogous to the first and second amounts as described in instant claim 5.
Therefore, claims 3-6 and 37 are prima facie obvious over Jiang in view of Drew and Golonka.
Claims 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al. (Cancer Discovery, 2020) in view of Kincaid et al. (WO 2019/140201 A1).
Regarding claims 7-9, Jiang teaches the methods of claims 1-2, 11-12, 33-34, 38-39, and 171 as described above. Jiang also performed their methods utilizing DNA from pregnant subjects, to differentiate fetal specific sequences from shared sequences. End motifs were found to differ between fetal and maternal DNA, where the CCCA end motif frequency was higher in fetuses, and the overall motif diversity score was lower (Figure 4D and E). However, the reference does not teach the use of fetal or placenta cells, nor the detection of a fetal or maternal abnormality.
Kincaid teaches methods and compositions for analyzing nucleic acids and preparing nucleic acid libraries (page 1, para. 2). These methods involve finding sequence reads (e.g. page 10, para. 1 and page 22, para. 1), and include end sequences (overhangs are at the end of nucleic acid sequences; page 22, para. 1, page 14, para. 5, and Figure 15). Overhang features may specifically be quantified, and a variety of features can be measured (page 84, para. 3-4 through page 87, para. 1). Overhang features can be used to determine the level of disease present in a sample (page 86, para. 2). Overhang features can include a classification of overhang type, including a 5’ overhang, 3’ overhang, or blunt end (page 85, lines 30-31). This reference also teaches the use of cell-free DNA from a tissue sample (page 43, para. 1). Kincaid also notes that overhang profiles can be developed for particular nucleases to determine the types of ends (e.g. blunt or overhang) that they produce (page 87, para. 2). This reference examined the digestion preferences of DNase I and found that preferred end signatures did exist (page 132, paras. 1-2). Kincaid teaches that the subject can be a pregnant female, and that placental cells can be detected in plasma (combinations of the samples listed can be used; page 42, para. 4 through page 43, para. 1). Fetal cells can specifically also be used, and fetal aneuploidies can be examined (page 43, para. 1).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Kincaid to inform the method of Jiang. Specifically, Kincaid teaches an overall similar method, where end sequences produced by particular nucleases are examined in relation to certain conditions. Kincaid specifically notes that their methods can examine fetal aneuploidies utilizing fetal cells and placental tissue. In thinking about these teachings in the context of the method of Jiang, which already suggests end motif differences between fetal and maternal DNA, the ordinary artisan would arrive at utilizing the full methods of Jiang concerning HCC to examine fetal aneuploidies. This would involve first testing the fetal and placental tissues for nuclease expression related to aneuploidies, then taking plasma DNA, separating it based on fetal and maternal DNA, and performing end motif analysis on aneuploidy and non-aneuploidy samples to determine if significant differences exist. This would then create a non-invasive way to determine if a fetus has an aneuploidy, which may affect pregnancy care and treatment, as well as allow for better preparations for newborn care upon birth. There would be a reasonable expectation of success as the overall end motif analyses of Jiang would be unchanged, the tissues and disorder focused on would simply be different.
Thus, claims 7-9 are prima facie obvious over Jiang in view of Kincaid.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al. (Cancer Discovery, 2020) in view of Serpas et al. (PNAS, 2019).
Regarding claim 10, Jiang teaches the methods of claims 1-2, 11-12, 33-34, 38-39, and 171, as described above. However, the reference does not directly teach analyzing organisms other than humans, though mouse studies are mentioned (e.g. page 665, column 2, para. 1). These mouse studies appear to inform the conclusions drawn by Jiang (page 671, column 1, paras. 2-4).
Serpas teaches DNASE1L3, and notes that this nuclease preferentially creates particular end motifs in nucleic acid fragments, including the end motif CCCA (Abstract and Table 1). This was done via a comparison of end motifs in wildtype mice versus mice that did not expression DNASE1L3 (Table 1). Serpas also notes that when DNASE1L3 is not expressed, autoimmune conditions can result in humans and mice (Abstract).
It is noted that Serpas is the reference cited throughout the parts of Jiang mentioned above that discuss mice (see reference 16 of Jiang). Thus, Jiang already used the results of Serpas as preliminary data to inform their human studies. Utilizing mouse studies before examining humans allows for easier development of protocols, and provides a baseline of expectations before beginning work with humans. Mice can also be more easily handled and examined than humans, allowing for more manipulative studies. As Serpas was already used to inform Jiang, it would be prima facie obvious that Serpas could also be used to inform the teachings of Jiang used to reject claim 1 above.
Thus, claim 10 is prima facie obvious over Jiang in view of Serpas.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al. (Cancer Discovery, 2020) in view of Maida et al. (World J Gastroenterol, 2014).
Regarding claim 13, Jiang teaches the methods of claims 1-2, 11-12, 33-34, 38-39, and 171, as described above. However, Jiang does not discuss particular stages of HCC.
Maida reviews various staging systems and prognoses for HCC (Abstract). Table 1 shows the staging systems. The BCLC system in particular breaks stages down from very early to end stage, where the treatment and care provided differ greatly between stages (page 4144, “BCLC staging classification”), and survival can be poor as stages progress (page 4142, column 1, paras. 1-4).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Maida to inform the method of Jiang. Specifically, Maida teaches that different stages of HCC are associated with different outcomes and require different care, highlighting the need to specifically identify the particular stage an HCC patient may have. Jiang already teaches the use of liver tumor sampling in their measures of DNASEIL3 expression (e.g. Figure 2A), and so these samples could also be used to identify the specific stage of HCC a patient may have. If the method of Jiang was then performed on plasma DNA from patients with various stages of HCC, motif frequencies and diversity scores could be generated for particular stages and compared to non-cancerous samples. This would then allow for not only a non-invasive way to test for HCC generally, but also to test for a particular stage of HCC. The ordinary artisan would recognize the utility in this, as it would provide an early and easy indicator of disease progression, which would determine the urgency of treatment/further testing, as well as the most effective treatment path for a patient. There would be a reasonable expectation of success as Maida teaches that methods of staging HCC tumors are globally ubiquitous and thus would be well-known to the ordinary artisan.
Thus, claim 13 is prima facie obvious over Jiang in view of Maida.
Claims 14-15 are rejected under 35 U.S.C. 103 as being unpatentable over Jiang et al. (Cancer Discovery, 2020) in view of Al-Mayouf et al. (Nature Genetics, 2011).
Regarding claims 14-15, Jiang teaches the methods of claims 1-2, 11-12, 33-34, 38-39, and 171, as described above. However, Jiang does not link DNAS1L3 to an auto-immune disorder.
Al-Mayouf teaches the discovery of an autosomal recessive form of systemic lupus erythematosus (SLE), involving a null mutation in the DNASEIL3 gene (Abstract). SLE generally is thought to result in a reduced ability to clear DNA from apoptotic cells, and in patients with it there is a deletion in the DNASE1L3 gene (page 1, column 2). When one or two copies of this mutant gene are present, there is either no DNASE1L3 activity or no detectible DNASE1L3 transcript in lymphoblasts and other tissues (page 2, column 2, para. 2).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Al-Mayouf as reason to take and analyze samples from subjects with auto-immune diso