DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . Applicant’s amendment filed 10/30/2025 is acknowledged. Claims 1 and 2 have been amended. Claims 16-19 have been canceled. Claims 1-15 and 20-30 are pending and under examination.
All of the amendment and arguments have been thoroughly reviewed and considered. Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims.
This action is made Final.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action
Previous Rejections
Status of claims:
-The objection to the drawings is withdrawn in view of Applicant’s replacement drawings.
-The claim objection is withdrawn in view of Applicant’s amendment of the claims.
-The claim rejection under 35 USC 101 directed to the claims 1-30 is withdrawn in view of Applicant’s amendment of the claims.
-The prior art rejection under 35 USC 103 directed to the claims 1-30 is withdrawn in view of Applicant’s amendment of the claims.
-The provisional double patenting rejections are maintained but modified to address Applicant’s amendment.
Double Patenting
3. The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
4. Claims 1-15 and 20-23 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-22 of copending Application No.18/706,482 {Application ‘482}.
An obviousness-type double patenting rejection is appropriate where the conflicting claims are not identical, but an examined application claim is not patentably distinct from the reference claim(s) because the examined claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F. 2d 887, 225 USPQ 645 (fed. Cir. 1985).
Although the claims at issue are not identical, they are not patentably distinct from each other because both the claims 1-15 and 20-23 of the instant invention and the claims 1-22 of copending application ‘482 are drawn to a method for detecting RAS pathway mutations in a subject, the method comprising: obtaining a biological sample from the subject; isolating nucleic acids from the biological sample and analyzing the expression level of the RNA compared to a control sample indicates that the subject has a RAS pathway mutation (claim 1 of instant invention and claim 1 of copending application ‘482). The claims 2-22 of copending application ‘482 embodies the limitations of the claims 1-15 and 20-23 of the instant invention.
The claims of copending application ‘482 differs from the claims 1-15 and 20-23 of the instant invention in that they are broader in scope and comprise of overlapping subject matter. The claims of the copending application and the instant application clearly comprise of a species/genus relationship. As the court stated in In re Goodman, 29 USPQ2d 2010 (CAFC 1993), “a second application-- "containing a broader claim, more generical in its character than the specific claim in the prior patent"--typically cannot support an independent valid patent. Miller, 151, U.S. at 198; See Stanley, 214 F.2d at 153. Thus, the generic invention, as noted above is "anticipated" by the species of the patented invention. Cf., Titanium metal corp. v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985) (holding that an earlier species disclosure in the prior art defeats any generic claims). This court's predecessor has held that, without a terminal disclaimer, the species claims preclude issuance of the generical application. "In re Van Ornum, 686 F.2d 937, 944, 214 USPQ 761, 767 (CCPA 1982); Schneller, 397 F.2d at 354".
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to Arguments
5. Applicant traverses the rejection on the following grounds: Applicant states that MPEP states that if a provisional non-statutory double patenting rejection the only rejection remaining in an application having the earlier patent term filing date, the examiner should withdraw the rejection in the application having the earlier patent term filing date and permit the application issue as a patent. Applicant states that the present application has the earlier patent term filing date and the co-pending application has a later patent term filing date. Applicant believes the response should address all other rejection leaving the present rejection the only remaining one and therefore the rejection should be withdrawn.
6. All of the arguments have been thoroughly reviewed and considered but are not found persuasive for the reasons that follow: The examiner acknowledges Applicant arguments but notes that since it cannot be determined which of the applications will be patented first and since this is not the only remaining issue in this application and Applicant has not filed a proper terminal disclaimer, the double patenting rejection is maintained.
Claim Rejections - 35 USC § 101
7. 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.
8. Claims 1-15 and 23-30 rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (i.e., a law of nature, a natural phenomenon, or an abstract idea) without significantly more. The claims recite method steps that appear to be directed to an abstract idea. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception for reasons that follows.
The independent claim 1 is directed a method for detecting a RAS pathway mutation in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) isolating nucleic acids from the biological sample; and (c) analyzing the expression level of noncoding RNAs in the nucleic acids in conjunction with a corresponding reference level in a control sample, wherein a differential expression level of the noncoding RNAs compared to the corresponding reference level in the control sample indicates that the subject has a RAS pathway mutation.
The dependent claims 2-15 and 20-30 embodies the limitations of the independent claim 1. See claims made of record 9/9/2022.
According to the Updates to interim Eligibility Guidance, an initial two step analysis is required for determining statutory eligibility. Step 1 requires a determination of whether the claims are directed to a process, machine, manufacture, or a composition of matter. In the instant case, the Step 1 requirement is satisfied as the claims are directed towards a process/machine. The Step 2 analysis is a two-part analysis. Step 2A and Step 2B, with the first part Step 2A requiring a determination of whether the claims are directed towards a judicial exception, i.e. a law of nature, natural phenomenon, or an abstract idea.
In the instant case, the claims are drawn to the abstract idea in the recitation of the following steps: “analyzing the expression level of noncoding RNAs in the nucleic acids in conjunction with a corresponding reference in a control sample (claim 1)”, “aligning the one or more sequencing reads…..” (claim 1) “comparing differential expression levels to a corresponding reference level” (claim 1), “analyzing the expression level of a gene involved in the interferon (IFN) alpha”, IFN gamma or KRAB-Zn Finger response” (claim 8), and “analyzing the expression level of noncoding RNAs transcribed from transposable elements” (claim 10).
As such, the instant claims are drawn to abstract processes that only collect/gather, manipulate or calculate data and/or by the use of general computer for performing such calculations and therefore, are not directed to statutory subject matter. Therefore, the result of Step 2A analysis is that the claims are directed towards a judicial exception, i.e. an abstract idea. The step recited therein in the claims merely encompass looking at data or algorithms or mathematical calculations to obtain sequence information. Additionally, the steps such as e.g., determining data, aligning data, collecting data and/or comparing data can additionally be interpreted as step encompassing mental analyses and/or reading data on paper.
The Court has made clear if a claim is directed essentially to a method of calculating, using a mathematical formula, even if the solution is for a specific purpose, the claimed method is non-statutory. In other words, patenting abstract ideas cannot be circumvented by attempting to limit the use [the idea] to a particular technological environment. In the instant case, performing general method steps to detect a Ras pathway mutation by analyzing data and comparing results is considered an abstract idea. The recitation of these steps in the method claims do not offer a meaningful limitation beyond generally linking “the use of the method to a particular technological environment,’ that is, implementation well-known general techniques for gathering and analyzing data.” see Alice Corp v. CLS Bank Int’l 573 U.S. (2014).
The second part, Step 2B of the two-step analysis is to determine whether any element or combination of elements, in the claim is sufficient to ensure that the claim as a whole amount to significantly more than the judicial exception.
In this case, the claims also recite measuring the expression level by performing sequencing, obtaining one or more sequencing reads, aligning the sequence reads to repetitive sequences.
The claims additionally broadly recite the step of administering to the subject one or more anticancer agents, and isolating extracellular vesicles from the sample on the basis of size wherein the size is about 150 nm or about 213 nm in diameter (claims 24-30).
These steps are recited at a high level of generality such that they represent well understood, routine and conventional activity undertaken by those in the associated technology as evidence by e.g., Kalluri et al (US 10598665, March 24, 2020) isolating nucleic acid sequencing and immunoblotting techniques (See col. 6-7, 13-15), administering anti-cancer agents (col. 18-21), and isolating extracellular vesicles based on size (col. 11-14) and Reggiardo et al (https://www.biorxiv.org/content/10.1101/2020.11.04.367771v1.full.pdf+html, November 4, 2020, pages 1-38, citation made of record in prior Office action) which teaches sequencing steps which encompasses steps of performing sequencing comprising obtaining sequencing reads on noncoding RNAs and aligning sequencing reads of the of the noncoding RNAs to repetitive sequences in human genome (see pages 12, 15, 18-19) . Further these steps do not provide a physical or an improved contribution to the judicial exception.
The Courts have made clear “essentially, appending conventional steps or elements, specified at a high level of generality, to laws of nature, natural phenomena, and abstract ideas cannot make those laws, phenomena, and ideas patent-eligible. The Supreme Court also indicated that any additional steps that simply are routine and conventional in the art are insufficient to transform an otherwise patent-ineligible process. Thus, the claims 1-30 are rejected as ineligible subject matter under 35 USC 101.
Response to Arguments
9. Applicant traverses the rejections on the following grounds: Applicant cites the clams as amended and states that claim goes beyond and abstract idea or any other judicial exception. Applicant state that the Supreme Court and Federal Circuit have identified a number of considerations as relevant to the evaluation under 35 USC 101. Applicant states in this case the claims as a whole improves upon the understanding of how the noncoding transcriptome contributes to cancer formation and their potential utility as an RNA-based liquid biopsy biomarker for diagnosing RNA driven cancers and thus integrates the recited judicial exception into practical application.
10. All of the amendment and arguments have thoroughly reviewed and considered but are not found persuasive for the reasons that follow: In response to Applicant’s arguments above, it appears Applicant is attempting to argue the claims integrate the recited judicial exception into a practical application that is an improvement to technology under step 2A, Prong 2. MPEP 2106.05(a) set forth:
An indication that the claimed invention provides an improvement can include a discussion in the specification that identifies a technical problem and explains the details of an unconventional technical solution expressed in the claim, or identifies technical improvements realized by the claim over the prior art.
It is important to note, the judicial exception alone cannot provide the improvement. The improvement can be provided by one or more additional elements. See the discussion of Diamond v. Diehr, 450 U.S. 175, 187 and 191-92, 209 USPQ 1, 10 (1981)) in subsection II, below. In addition, the improvement can be provided by the additional element(s) in combination with the recited judicial exception. See MPEP § 2106.04(d) (discussing Finjan, Inc. v. Blue Coat Sys., Inc., 879 F.3d 1299, 1303-04, 125 USPQ2d 1282, 1285-87 (Fed. Cir. 2018)). Thus, it is important for examiners to analyze the claim as a whole when determining whether the claim provides an improvement to the functioning of computers or an improvement to other technology or technical field.
Consideration of improvements is relevant to the eligibility analysis regardless of the technology of the claimed invention. That is, the consideration applies equally whether it is a computer-implemented invention, an invention in the life sciences, or any other technology. See, e.g., Rapid Litigation Management v. CellzDirect, Inc., 827 F.3d 1042, 119 USPQ2d 1370 (Fed. Cir. 2016), in which the court noted that a claimed process for preserving hepatocytes could be eligible as an improvement to technology because the claim achieved a new and improved way for preserving hepatocyte cells for later use, even though the claim is based on the discovery of something natural. Notably, the court did not distinguish between the types of technology when determining the invention improved technology. However, it is important to keep in mind that an improvement in the abstract idea itself (e.g. a recited fundamental economic concept) is not an improvement in technology. For example, in Trading Technologies Int’l v. IBG, 921 F.3d 1084, 1093-94, 2019 USPQ2d 138290 (Fed. Cir. 2019), the court determined that the claimed user interface simply provided a trader with more information to facilitate market trades, which improved the business process of market trading but did not improve computers or technology.
In the instant claims, the only additional elements recited are isolating the nucleic performing sequencing on the isolated nucleic acid and aligning the one or more sequencing reads of noncoding RNAs to repetitive sequences in a human genome.
As discussed above, the technical improvements by the claim must be realized over the prior art and must be provided by either the additional elements or the additional elements in conjunction with the recited judicial exception. Relative to the prior art, the additional elements sequencing and aligning were already readily available, as evidenced by Applicant’s own specification (see page 36-37) and other prior art references (see above rejection, especially Reggiardo which teaches similar techniques of sequencing as recited in the instant invention). This indicates that the any improvements over the prior art lie purely in the judicial exception and there is no indication that the judicial exception is improving the assay steps to help them perform better. Instead, the instant claims recite applying a technique already available in the prior art to gather data needed as input for the particular judicial exception. Therefore, these limitations are more akin to insignificant extra-solution activity rather than additional elements that integrate the judicial exception to result in an improvement to technology.
NEW GROUNDS OF REJECTION
THE NEW GROUND(S) OF REJECTIONS WERE NECESSITATED BY APPLICANT’S AMENDMENTS OF THE CLAIMS:
NOTE: The following are new grounds of rejections necessitated by Applicant's amendments. Although the claims were previously rejected as being anticipated and/or unpatentable over the same reference(s), Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action. It is noted that, to the extent that they apply to the present rejection; Applicant's arguments are addressed following the rejection.
Claim Rejections - 35 USC § 103
11. 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.
12. 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.
13. 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.
14. Claim(s) 1- 15 and 20-30 is/are rejected under 35 U.S.C.103 as being unpatentable over Kalluri et al {Kalluri, used interchangeably herein} (US 10598665, March 24, 2020) in view of Reggiardo et al {Reggiardo, used interchangeably herein} (BioRxiv, https://www.biorxiv.org/content/10.1101/2020.11.04.367771v1.full.pdf+html, November 4, 2020, pages 1-38).
Regarding claims 1, 4-7, Kallluri teaches a method for detecting a RAS pathway mutation in a subject, the method comprising: identifying a cancer biomarker in a subject, wherein the cancer biomarker may a KRAS mutation (col. 3, lines 7-8; col. 4, lines 1-4), the method comprising obtaining a biological sample from the sample (col. 2, lines 44); isolating nucleic acids from the biological sample (col. 4, lines 39-41; col. 9-12); and analyzing the expression level of extracellular RNAs in the nucleic acids, wherein a differential expression level of the extracellular RNAs compared to a control sample indicates that the subject has a RAS pathway mutations (col. 2-7, 12-15, col. 45, lines 12-15; col. 46, lines 4-17; col. 47, lines 25-27, 40-41, Table 2; Figures 1, 2, 5, 11, 13, 19; see also Examples 2 and 3). Kalluri teaches wherein non-coding RNAs are analyzed (col. 40, lines 21-38). Kalluri further teaches wherein cancer is a RAS mutant cancer and wherein the RAS mutant cancer is a lung, cancer, pancreatic cancer, colorectal cancer, or melanoma (col. 2 line 2 to col. 3, line 25).
Regarding claim 2, Kalluri teaches where the RAS pathway mutation is in KRAS (Fig 2A-E, Fig. 11F, Fig. 12A-F, Fig. 19), EGFR (col. 12, line 21).
Regarding claim 3, Kalluri teaches wherein the biological sample comprises extracellular vesicles isolated from biofluids from the subject (abstract, col., 1, line 60 to col. 2, first full paragraph).
Regarding claim 8, Kalluri teaches wherein the method further comprises analyzing the expression level of a gene involved in the interferon IFN gamma or Zn Finger response (col. 21, lines 45 – 49, Table 1).
Regarding claims 20-23, Kalluri teaches wherein the biological sample is a blood sample, a urine sample, a saliva sample or a tissue sample and wherein the subject is a human (col. 2, lines 49 to col. 3, lines 42; col. 13 first full paragraph).
Regarding claim 24-26, Kalluri teaches further comprising administering to the subject one or more anticancer agents, wherein the anticancer agent is an inhibitor of a RAS pathway gene (col. 3-7, col. 12).
Regarding claims 27-30, Kalluri teaches further comprising isolating extracellular vesicles from the sample on the basis of size wherein the size is from about 40 – 150 nm (col. 11 line 64; col. 13, line 64 to col. 14, 11). Kalluri does not teach wherein the vesicles are from about 150 nm or about 213 nm in diameter. However, MPEP states "[A] prior art reference that discloses a range encompassing a somewhat narrower claimed range is sufficient to establish a prima facie case of obviousness." In re Peterson, 315 F.3d 1325, 1330, 65 USPQ2d 1379, 1382-83 (Fed. Cir. 2003). See also In re Harris, 409 F.3d 1339, 74 USPQ2d 1951 (Fed. Cir. 2005).
While, Kalluri teaches number aspects of the claimed invention including analyzing differential expression levels of RNA, analyzing increase, decrease or lack of change in cancer derived exosomes to detect cancer in a sample and response to therapy, the reference does not teach in the sequencing step, aligning the one or more sequencing reads of the noncoding RNSA to repetitive sequences in a human genome, analyzing expression level of noncoding RNA transcribed from transposable elements or wherein an increase in the expression level of the gene involved in the IFN alpha, IFN-gamma, or a decrease in the expression level of a gene involved in the KRAB-Zn finger response relative to a corresponding reference level for the gene in the control sample from the control subject indicates that the subject has cancer.
Regarding claims 1-15 and 23-30, Reggiardo teaches a method of characterizing noncoding RNAs, transposable-elements derived repetitive noncoding RNA and extracellular RNA using human sample that have a constitutively active mutant KRAS allele. Reggiardo teaches that they show that oncogenic KRAS induces cell-intrinsic interferon (IFN)-stimulated gene (ISG) signatures through epigenetic and RNA-mediated mechanisms, and that KRAB zinc finger (KZNF) genes that repress repetitive noncoding RNA loci are globally down-regulated both in vitro and in vivo in lung adenocarcinoma patients with activating mutations in KRAS (last paragraph at page 3). Reggiardo states that their data revealed that significant upregulation and extracellular release of repetitive noncoding RNAs and ISGs are early transcriptomic signatures of mutant kRAS signaling (see page 4, lines 1-2). Regiardo et al teach to explore the biological pathways that are perturbed by oncogenic RAS signaling, we performed gene set enrichment analysis (GSEA) (15) using genes that were differentially expressed in our mutant KRAS human airway epithelial cells (AALE). GSEA revealed that the most significantly enriched pathway was the interferon (IFN) alpha response, while the third most enriched pathway was the IFN gamma response (Supplementary Figure 1c). We examined all known ISGs that were expressed in our mutant KRAS AALEs and found that most of these genes were significantly upregulated, comprising a 25-gene mutant KRAS ISG signature (Fig. 1a). We then compared our KRAS ISG signature to IFN alpha (INFa) and IFN gamma (IFNg) response genes. While the majority of KRAS ISG signature genes overlapped with the other 3 IFN-related signatures, several genes were unique to mutant KRAS cells (Fig. 1b). These results reveal that mutant KRAS signaling activates an intrinsic ISG response in transformed human epithelial cell samples.
Reggiardo teaches that they next investigated the molecular basis for intrinsic ISG signature activation in mutant KRAS AALE cells by analyzing the abundance of repetitive noncoding RNAs transcribed from TEs, which induce an IFN response in cancer cells when aberrantly expressed. The LINE-1 element L1MC4a, the Alu elements AluSx, AluSg, AluJo, AluY, and AluSz6, and the hAT-Charlie element MER20 were all significantly upregulated in mutant KRAS AALE cells (Fig. 3a), suggesting that oncogenic KRAS signaling induces an ISG signature in transformed lung cells through the activation of TE-derived noncoding RNAs. We examined TE expression heterogeneity in our single-cell RNA-seq data from mutant KRAS AALEs and did not observe substantial heterogeneity in ALU, LINE, MER, or LTR class TE expression (Fig. 3b), as well as for the specific TEs L1MC4a and AluSx (Fig. 3c). When we performed RNA editing analysis on the scRNA-seq data to look for Ato-I editing (21), however, we found that TE-derived double-stranded RNAs (dsRNAs) exhibited significantly lower levels of RNA editing in cluster 4 (Fig. 3d), even though TE RNA levels were similar across all of the scRNA-seq data clusters. Mutant KRAS cells with lower levels of RNA editing exhibited higher expression of the dsRNA sensors MDA5 and RIG-I, while all of the clusters showed relatively similar levels of PKR expression, with a slightly higher level of PKR expression in the cells with lower RNA editing (Fig. 3e). (see page 7, section “Mutant KRAS upregulates transposable element derived noncoding RNAs).
Reggiardo teaches to test whether extracellular RNAs that are released from mutant KRAS cells might also exhibit differential RNA editing, we isolated extracellular vesicles from the culture media of control and mutant KRAS AALEs. Extracellular vesicles isolated from mutant KRAS AALE cell culture media were comprised of two different sized classes of vesicles that were ~150nm and ~213nm in diameter, while vesicles from control AALE media were ~196nm in size (Supplementary Figure 2a). We performed RNA-seq and found that extracellular vesicles were enriched in lncRNAs when compared to the intracellular RNA composition (Supplementary Figure 2b). We also observed strong correlation between differentially expressed genes in intracellular and extracellular RNAseq data (Supplementary Figure 2c). Moreover, repetitive noncoding RNAs derived from Alu, ERV, and L1 elements were significantly enriched in extracellular vesicles (Supplementary Figure 2d) but did not exhibit differential RNA editing (Supplementary Figure 2e), suggesting that intrinsic KRAS ISG signatures in mutant KRAS AALEs are not significantly affected by TE-derived repetitive noncoding RNAs that are packaged into extracellular vesicles (page 8).
Reggiardo teach given the known roles of KRAB zinc-finger proteins (KZNFs) in TE silencing (24), we examined whether KZNFs were involved in TE regulation in mutant KRAS AALEs. When we examined the differential expression of KZNFs in mutant KRAS AALEs, we observed a broad and significant down-regulation of repressive KRAB domain-containing zinc- finger proteins (Fig. 4a) (Supplementary Figure 3a). Based on our ATAC-seq experiments which comprised of alignment quality control and psedoalignment of RNA-seq reads using Salmon (see pages, 15, 18 and 19), we determined that significantly down-regulated KZNF genes exhibited loss of open chromatin at their TSS and intronic regions (Fig. 4b), revealing that mutant KRAS signaling induces epigenetic silencing of many KZNF genes. Several significantly downregulated KZNFs, including ZNF90, ZNF736, and ZNF683, were enriched for motifs in their TSS regions for ETS and ELK transcription factors (Fig. 4c), which are known downstream effectors of the RAS signaling pathway together with AP-1 (FOS) (11) (Fig. 4d).
We then analyzed KZNF chromatin immunoprecipitation sequencing (ChIP-seq) data (24) using the University of California Santa Cruz (UCSC) Repeat Browser platform. AALEs bind to the consensus TE sequences of MER20 and L1MC4a elements (Supplementary Figure 3b), which are specifically and significantly upregulated in mutant KRAS AALEs (Fig. 3a). This suggests that suppression of these KZNFs via oncogenic RAS signaling leads to de-repression of TE-derived noncoding RNAs during cellular transformation. This model is supported by broad and significant down-regulation of these same KNZFs in vivo in mutant KRAS-driven lung adenocarcinomas (Supplementary Figure 3c). Riggiardo teaches that their studies suggest that oncogenic RAS signaling contributes to the early induction of intrinsic ISG signatures that are observed across many cancers and cancer cells lines with ADAR dependencies (page 10).
It would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have been motivated to incorporate differential expression analysis of transposable elements derived repetitive noncoding RNA in RAS pathway mutations based on sequence read information as taught by Reggiardo in the RAS pathway mutation detection method of Kalluri. The ordinary artisan would have been motivated to do for increase means of diagnosing RAS-driven cancers in early stages as suggested by Reggiardo.
Response to Arguments
15. Applicant traverses the rejections on the grounds that Kalluri et al does not teach analyzing the expression level of noncoding RNA in the nucleic acid in conjunction with a corresponding reference level in a control sample, wherein a differential expression level of the noncoding RNAs compared to the corresponding reference level in the control sample indicates that the subject has a RAS pathway mutation.
16. All of the arguments have been thoroughly reviewed and considered but are not found persuasive for the reasons that follows: The examiner acknowledges Applicant’s arguments but notes in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In this case, the secondary teaching of Reggiardo supplements for what is missing in the prior art of Kalluri by teaching epigenomic reprogramming of repetitive noncoding RNA and IFN-stimulated gens by mutant KRAS (see entire document). The combination of the cited prior art provides a reasonable case of obviousness and thus Applicant’s arguments are not found persuasive.
Conclusion
17. No claims are allowed. Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
18. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681