DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 06 October 2025 has been entered.
Status of the Claims
The claim set received 06 October 2025 has been entered into the application.
Claims 3, 7, 12, 16, 19, 22-24, and 28 are amended.
Claims 29-30 are new.
Claims 6 and 27 are cancelled.
Claims 1-2, 4-5, 9-11, 14-15, 17-18, and 20 previously are canceled.
Claim(s) 3, 7-8, 12-13, 16, 19, 21-26, and 28-30 are pending.
Election/Restrictions
The Applicant has elected Group II, claims 3-19 drawn to a system determining the state of tissue of interest in a subject based on principle component analysis. Election was made without traverse in the reply filed on 09 July 2024.
Priority
Acknowledgment is made of applicant’s claim for priority to U.S Provisional Application filed 27 January 2012. Application 17/187,298 is a continuation of abandoned application 16/373,996 filed 03 April 2019 which is a continuation of 15/377,894 (U.S Patent 10,287,632) filed 13 December 2019 which is a continuation of 14/861,650 (U.S Patent 10,240,200) filed 26 March 2019 which is a divisional of 13/752,131 (Abandoned) filed 28 January 20213 which claims priority benefit to U.S Provisional Application 61/591,642 filed 27 January 2012.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 08 October 2025 and 08 October 2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
It is noted that copies were not provided for NPL Cite No’s. 51 and 53.
Specification
The objection to the specification in the Office Action mailed 06 June 2025 is withdrawn in view of the amendments received 06 October 2025.
Claim Rejections - 35 USC § 112
35 USC § 112(a)
The rejection of claims 27-28 under 35 U.S.C § 112(a) in the Office Action mailed 06 June 2025 is withdrawn in view of the amendments received 06 October 2025.
35 USC § 112(b)
The rejection of claims 24 under 35 U.S.C § 112(b) in the Office Action mailed 06 June 2025 is withdrawn in view of the amendments received 06 October 2025.
Claim Rejections - 35 USC § 101
The instant rejection is maintained for reason for record in the Office Action mailed 06 June 2025 and modified in view of the amendments filed 06 October 2025. It is noted the amendments received 06 October 2025 necessitated new ground(s) of rejection.
The rejection of claims 6 and 27 under 35 U.S.C § 101 in the Office Action mailed 06 June 2025 is withdrawn in view of the amendments received 06 October 2025.
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 3, 7-8, 12-13, 16, 19, 21-26, and 28-30 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter.
Claims Analysis
Under broadest reasonable interpretation, the claims are direct towards a system for analyzing and evaluating nucleic acid data, more specifically, cell-free messenger ribonucleic acids (cf-mRNAs) for deconvolving tissue types.
Following the flowchart of the MPEP 2106
Step I - Process, Machine, Manufacture or Composition
Claims 3, 7-8, 12-13, 16, 19, 21-26, and 28-30 are drawn to a method, so a process.
2A Prong I - Identification of an Abstract Idea
Claim 1 recites
(d) wherein the collection of sequencing data comprises test fractional contributions of tissue types.
This step describes the sequencing data as comprising test fractional contributions.
(e) computer processing the collection of sequencing data wherein the computer processing comprises deconvoluting the test fractional contributions of tissues types from plasma derived from the test blood sample of the test subject against a reference cell-free transcriptome in a reference blood sample of a reference subject.
This step can be performed in the human mind by following instructions to deconvolute sequencing data into test fractional contributions of tissue types against a reference cell free transcriptome and is therefore an abstract idea. This step encompasses performing mathematical/statistical computations to deconvolute test fraction contribution of tissue types against a reference cell free transcriptome and is therefore an abstract idea. The step encompasses organizing and manipulating information (i.e., sequencing data) through mathematical correlations (i.e., deconvolution and/or test fractional contributions) and is therefore an abstract idea. Here, the claims take existing data (i.e., sequencing data) and manipulate the data using mathematical correlations by deconvolution to produce test fractional contributions of cells and/or tissue types. See MPEP 2106.04(a)(2)(I)(A)(iv). The term “test fractional contributions” is interpreted as proportion of cells of a specific tissue type(s).
wherein deconvolving the test fractional contributions of tissue types further comprises: (1) identifying a panel of tissue-associated transcripts; (2) determining total ribonucleic acid (RNA) in plasma derived from the test blood sample of the test subject; and (3) assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1), wherein the total RNA is considered a summation of the tissue-associated transcripts.
This step can be performed in the human mind by following instruction to (1) identify a panel of tissue-associated transcripts and (2) determine the total RNA of step (2) against the panel of tissue-associated transcripts of step (1) and observing, comparing, assessing, and evaluating the identified panel of tissue-associated transcripts and determined (i.e., summed) total RNA to deconvolve test fractional of tissues types and is therefore an abstract idea.
wherein the total RNA is considered a summation of the tissue-associated transcripts.
This step describes the total RNA as a summation of the tissue-associated transcription which encompasses mathematical concepts of summation with respect to summing the tissue-associated transcription to yield a total RNA which reads on abstract ideas/mathematical concepts.
Claims 3, 7-8, 12-13, 16, 19, 21-26, and 28 are further drawn to limitations that describe the abstract ideas of claim 3 and are therefore also abstract ideas.
2A Prong II - Consideration of Practical Application
Claims 3 do not recite any additional element which integrates the recited judicial exception into a practical application. Here, in the instant case, the claims merely set forth a method of data analysis for analyzing sequence data to produce test fraction contributions of cells and/or tissue types for deconvoluting cell into tissue types. Such a result only produces information (i.e., deconvolved test fraction contribution from sequencing data) and does not provide for a practical application in the real-world realm of physical things and acts, i.e., the claims do not utilize the data generated by the judicial exception to affect any type of change. See MPEP 2106.04(a)(2)(A)(iv). Therefore, the claims do not utilize the isolated cfRNA, obtained cDNA, amplified cDNA, sequenced data, and deconvoluted test fractional contributions and the abstract ideas to "transform the nature of the claim” in order to construct a practical application such as treating a subject, transformation of matter, or improving upon an existing technology.
This judicial exception is not integrated into a practical application because the claims do not meet any of the following criteria:
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.
Step 2B: Consideration of Additional Elements and Significantly More
The claimed method also recites "additional elements" that are not limitations drawn to an abstract idea.
The recited additional element of isolating nucleic acids from a blood sample of claim 3 step (a) does not add significantly more than the recited judicial because isolating cell-free cfRNA from blood and/or tissues that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II)(i). To provide evidence of conventionality, Swarup et al. (Swarup) teaches a min-review of using circulating cell-free nucleic acids for detection of human disease [title]. Swarup teaches a schematic of various pathways which cell free nucleic acids are released into the system [page 797 fig 1] (FEBS letters, 2007-03, Vol.581 (5), p.795-799). To provide further conventionality. Kurn et al. (Kurn) teaches using cell-free RNA with respect to compositions and methods for whole transcriptome analysis [claims 14, 31, and 48] (US 2011/0189679). To provide further evidence of conventionality, Kroh et al. (Kroh) teaches analysis of circulating microRNA biomarkers [abstract]. Kroh teaches analyzing microRNA in plasma and serum [page 298 right section 2.2.1]. Kroh teaches plasma and serum are acceptable types of specimens for circulating miRNA analysis [page 299 section 2.2.3] (Methods (San Diego, Calif.), 2010-04, Vol.50 (4), p.298-301).
The recited additional element of isolating cfRNA by adding cell-membrane stabilizing agent of claim 3 step (a) does not add significantly more to the recited judicial exception because adding membrane stabilizers for further collecting nucleic acid data that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II)(i, iii, v, vii). Adding cell-membrane stabilizers to blood samples to prevent cell degradation and preserve the integrity of cells and their components for accurate testing and analysis is well known. To provide evidence of conventionality, Dhallan et al. (Dhallan) discloses collecting free fetal DNA (cfDNa) and that agents were added to samples to inhibit cell lysis such as membrane stabilizers, a cross-linker, or a cell lysis inhibitor [disclosure page 5 right col. para. 0057] to samples including blood samples [para. 0058]. Dhallan discloses “membrane stabilizers agents such has added to the sample including but not limited to aldehydes, urea formaldehyde, phenol formaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, Vitamin E, and Vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, Sucrose, astaxanthin, glucose, amitriptyline, isomer A hopane tetral phenylacetate, isomer Bhopane tetral phenylacetate, citicoline,inositol, Vitamin B, Vitamin B complex, cholesterol, hemisuccinate, Sorbitol, calcium, coenzyme Q, ubiquinone, Vitamin K, Vitamin K complex, menaquinone, zonegran, Zinc, ginkgo biloba extract, diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine, tegretol, PABA, disodium cromglycate, nedocromil Sodium, phenyloin, Zinc citrate, mexitil, dilantin, Sodium hyaluronate, or polaxamer 188.” [disclosure page 9 left col para. 0078] [Instant specification page 11 first paragraph] (US Patent Pub 2004/0137470, Patent Pub Date: 15 July 2004).
To provide further conventionality of isolating cfRNA by adding cell-membrane stabilizing agent, Quake et al. (Quake) discloses using cell membrane stabilizers or impeding cell lysis [page 6 left col para. 0071]. Quake discloses different chemicals that can be used as cell membrane stabilizers or cell lysis impeders such as “aldehydes, urea formaldehyde, phenol formaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, vitamin E, and vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, and coenzyme Q, for example [disclosure page 6 para. 0073]. Quake discloses that “while the present description refers to DNA, fetal RNA found in maternal maybe analyzed as well [page 5 right col. para. 0063] (US Patent Pub 2007/020525, Patent Pub Date: 30 August 2007).
To provide further conventionality of using cell membrane stabilizer and/or agents, Quake et al. (Quake 2009) discloses genetic material maybe DNA or RNA, preferably mRNA [disclosure page 2 right col para. 0026]. Quake 2009 discloses agents for stabilizing cell membrane or cell lysis inhibitors such as Vitamin E, DMAE, glucose, aldehydes, Dilantin, for example [disclosure page 6 left col para. 0065] (Quake 2009: US Patent Pub 2009/0170113, Patent Pub Date: 02 July 2009).
The recited additional element of obtaining nucleic acids of claim 3 step (b) does not add significantly more than the recited judicial because reverse transcribing RNA to obtain cDNA data that is subsequently amplified is well-known and conventional. See MPEP 2106.05(d)(II) (v and viii). To provide conventionality of generating/obtaining double stranded cDNA products, Kurn teaches optionally generating double stranded cDNA products form reversed transcribed products [claims 18 and 34] [disclosure paragraphs [0028-0032]]. To provide further conventionality of transcribing RNA to obtain cDNA, Klickstein et al. (Klickstein) teach a general construction of cDNA libraries from mRNA (Klickstein: Current Protocols in Molecular Biology (1995) 5.5.1-5.5.14). To provide further conventionality of reverse transcribing RNA to cDNA, Qiagen 2011 teaches selective conversion of mature miRNAs into cDNA (i.e., double stranded DNA) [page 11 figure 2]. Qiagen 2011 teaches Simultaneous conversion of all RNA species into cDNA (i.e., double stranded DNA) in miScript HiFlex Buffer [page 12 figure 3] (Qiagen Sample and Assay Technologies: miScript PCR System Handbook 2011). To provide further conventionality of reverse transcribing RNA to cDNA, Invitrogen 2001 teaches an outline of procedures for cDNA synthesis [page 2 figure 1]. Invitrogen 2001 teaches converting mRNA to ds cDNA products [page 6 fig 3] (Invitrogen cDNA Synthesis System 2001). To provide further evidence of conventionality, Kroh teaches a protocol for reverse-transcription of miRNA using a TaqMan miRNA reverse transcription kit [page 300 left col section 2.6.1]. To provide further evidence of conventionality, Kurn teaches reverse using transcription of cell free RNA to generate double stranded cDNA products [claims 1 steps (b)-(c) and 15].
The recited additional element of amplifying nucleic acids of claim 3 step (c) does not add significantly more than the recited judicial because amplifying cDNA to produce dsDNA that is subsequently sequenced is well-known and conventional. See MPEP 2106.05(d)(II) (vii). To provide conventionality of amplifying cDNA to produce dsDNA, Kurn teaches amplifying double-stranded cDNA [claims 18 and 34]. Kurn further teaches that reversed transcribed product are amplified before being sequenced [claims 4-5].
The recited additional element of sequencing nucleic acids of claim 3 step (d) does not add significantly more than the recited judicial because sequencing cDNA to obtain sequencing data that is subsequently analyzed by abstract idea(s) is well-known and conventional. See MPEP 2106.05(d)(II) (v and vii). To provide evidence of conventionality, Kurn teaches sequencing of amplified reverse transcribed products [claims 4-5].
The additional element of using computer process, components, and equipment of claim 3 step (e) does not add significantly more than the recited judicial exception because using computer processes and equipment to analyze sequence data and process abstract ideas is well-known and conventional. See MPEP 2106.05(b) and 2106.05(d).
The recited additional element of sequencing nucleic acids of claim 26 does not add significantly more than the recited judicial because next generation sequencing to obtain sequencing data that is subsequently analyzed by abstract idea(s) is well-known and conventional. See MPEP 2106.05(d)(II) (ii, v, and vii).
The recited additional element of producing cDNA library of claim 29 does not add more than the recited judicial exception because producing cDNA libraries that are used to produce sequencing data that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II) (ii, v, and vii).
The recited additional element of performing PCR on the cDNA of claim 30 does not add more than the recited judicial exception because performing on cDNA to produce nucleic acid sequences that are further sequenced to produce sequencing data that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II) (ii, v, and vii).
In conclusion, and when viewed as a whole, these additional claim element(s) do not provide meaningful limitation(s) to transform the abstract idea recited in the instantly presented claims into a patent eligible application of the abstract idea such that the claim(s) amounts to significantly more than the abstract idea itself. Therefore, the claim(s) are rejected under 35 U.S.C. 101 as being directed to non-statutory subject matter.
Response to Arguments
Applicant’s arguments, filed 06 October 2025, have been fully considered but the rejection is maintained. Furthermore, upon further consideration, a new ground(s) of rejection is made in view of amendments received 06 October 2025.
The Applicant states “the instant claims are patent-eligible, for at least the reason that, under the Step 2B analysis of the 2019 Revised Patent Subject Matter Eligibility Guidance (2019 PEG), the instant claims as a whole provide an inventive concept that amounts to significantly more than the alleged judicial exception. For example, claim 3 recites the following ordered combination of limitations, which was not well-understood, routine, or conventional as of the effective filing date of the Application”. The Applicant points to claim 3 steps (d) for clarification. The Applicant points to the MPEP 2106.05, BASCOM and Rapid Litig for guidance [remarks, pages 8-9]. The Applicant states “ Particularly, Applicant submits that, in the field of molecular assessment of tissue types, it was not well-understood, routine, or conventional as of the effective filing date of the Application for one of ordinary skill in the art to perform the following ordered combination of additional limitations beyond the alleged judicial exception: "(a) isolating cell-free ribonucleic acid (cfRNA) from plasma derived from a test blood sample of a test subject, wherein the cfRNA comprises cell-free messenger RNA, wherein isolating the cfRNA comprises adding a cell membrane stabilizing agent to the test blood sample; (b) reverse transcribing the cf RNA to obtain complementary deoxyribonucleic acid (cDNA); (c) amplifying the cDNA to produce double-stranded cDNA amplification products; [and] (d) performing a sequencing assay on the double-stranded cDNA amplification products" (emphases added).” The Applicant points to the instant specification [0055 and 0116] for clarification. The Applicant states “For example, Applicant submits that the claimed methods-including the additional element of "adding a cell-membrane stabilizing agent to the test blood sample" provide an improvement over other methods in the field of molecular assessment of tissue types.” The Applicant states “the claimed methods advantageously include adding a cell-membrane stabilizing agent to a test blood sample in order to ensure that cfRNA released from apoptotic cells of the test subject is subjected to molecular analysis, while limiting RNA produced ex-vivo of the test subject by cells that are lysed during the molecular analysis. Accordingly, the claimed invention recites additional limitations that amount to significantly more than the alleged judicial exception and therefore provide an inventive concept.” [remarks, page 9].
In response, claim 1 steps (a)-(d) encompass additional elements for performing clinical tests using cell-free RNA (cfRNA) for deconvolving tissue-associated transcription. Here, isolating cfRNA, using cell-membrane stabilizers during cell free nucleic acid collection, and reverse transcribing cfRNA, amplifying, and sequencing are routine and well-known for gathering nucleic acid data (i.e., cfRNA data/tissue associated transcripts) that is subsequently analyzed by the abstract ideas. Furthermore, the laboratory/clinical techniques of steps (a)-(d) encompasses laboratory techniques that are extra-solution activities i.e., collecting nucleic acid sequence data and determining the level of a biomarker(s) (i.e., mRNA) in blood. See MPEP 2106.05(g)(vi) “Determining the level of biomarker in blood.” Thus, consequently, adding physical steps such as isolating cfRNA using cell-membrane stabilizers does not provide an inventive concepts because the mere addition of physical steps (i.e., the steps of DNA (i.e., RNA) isolation using cell-membrane stabilizers, reverse transcribing, amplification, and sequencing) is not "sufficient" to render a claims patent eligible. See Genetic Technologies Ltd. v. Merial LLC, 818 F.3d 1369, 1377, 118 USPQ2d 1541, 1547 (Fed. Cir. 2016). Moreover, as noted in the 35 U.S.C § 101 rejection above, there are multiple references which disclose, recite, and teach the conventionality of isolating cells, using membrane stabilizers, reverse transcribing, and amplifying for sequencing nucleic acids. See MPEP 2106.05(d)(II)(i-iii, v, vii). It is noted that these step, regardless or order, encompass common scientific knowledge (i.e., common laboratory steps) utilized for collecting nucleic acid data from cells. For example, with respect to the ordered steps of claim 3 steps (a)-(d) being unconventional, it is noted the specification [page 10 top of page] discloses “methods of the invention involve isolating total RNA from a biological sample. Total RNA can be isolated from the biological sample using any methods known in the art.” Here, it appears claim 3 steps (a)-(d) can be performed by any method known in the art for collecting and processing RNA from a sample to yield a total RNA. As such, claim 3 steps (a)-(d) are conventional and therefore do not present unconventional methods (i.e., ordered steps) for processing RNA for deconvolving tissue types.
With respect to the ordered steps of Rapid Litig. Mgmt. v. CellzDirect, the claimed process was drawn to producing a ordered preparation of hepatocytes "capable of being frozen and thawed at least two times,'' comprising performing density gradient fractionation to separate viable and non-viable hepatocytes, recovering the viable hepatocytes, and cryopreserving the recovered viable hepatocytes which encompassed a combination of claimed elements that employed the natural discovery to create a new and improved way of preserving hepatocyte cells for later use. Whereas, the instant claims utilize conventional and tangential additional elements for collecting nucleic acid sequence data (i.e., RNA sequence data) for deconvolving test fractional contributions of tissue types.
With respect to BASCOM, the claimed invention is not relevant to the instant claims because BASCOM was found to be an inventive concept because the implementation of using generic computer network or internet components (i.e., special ISP server that receives requests for Internet content, which the ISP server then associates with a particular user and a particular filtering scheme and elements) in non-conventional and non-generic arrangement/order which provided a technical improvement. See MPEP 2106.05(a)(II)(ix) and (ii-iii). In contrast, the additional elements of the instant claimed invention are routine, well-known, and conventional laboratory elements for collecting, transcribing, amplifying, and sequencing cell free nucleic acids for obtaining nucleic sequence data. See MPEP 2106.05(d)(II)(i-iii, v, vii-viii) and MPEP 2106.05(g)(vi).
Additionally, with respect to using cell membrane stabilizers, Dhallan, Quake, and Quake 2009 disclose using cell membrane stabilizers for inhibiting lysis cells. Thus, using cell membrane stabilizer to prevent lysis of extraneous cells to prevent release of unwanted cellular RNA and/or DNA during plasma nucleic acid collection is well-known, routine, and conventional. Therefore, the instant claimed invention does not integrate the recited judicial exception into a practical application and does not recite additional limitations that amount to significantly more (i.e., inventive concept).
Furthermore, the claims do not provide an improvement because the claims are drawn to mere gathering and analyzing information (i.e., sequencing data) using conventional techniques (i.e., isolating of cell-free RNA (cfRNA), reverse-transcribing of cfRNA to obtain cDNA, amplifying the cDNA, and sequencing the cDNA) and displaying the result (i.e., deconvolved tissue types). See MPEP 2106.05(a)(II)(iii).
Therefore, the instant claimed invention is not patent eligible under 35 U.S.C § 101.
Claim Rejections - 35 USC § 103
The instant rejection is maintained for reason for record in the Office Action mailed 06 June 2025 and modified in view of the amendments filed 06 October 2025. It is noted the amendments received 06 October 2025 necessitated new ground(s) of rejection.
The rejection of claims 3, 22, 24, and 26 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Gong et al. (Cited in the Office Action mailed 10 October 2024) (PloS one, 2011-11, Vol.6 (11), p.e27156-e27156) in view of Kurn et al. (US2011/0189679 Pub: 04 August 2011) in view of Tani et al. (Cited in the Office Action mailed 10 October 2024) (Anticancer research, 2007-03, Vol.27 (2), p.1207-1212) in the Office Action mailed 06 June 2025 is withdrawn in view of the amendments received 06 October 2025.
The rejection of claims 12-13 and 16 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Gong in view of Kurn in view of Tani, as applied to claim 3, 22, 24, and 26 above, and in further view of McGill (Tutorial: RMA Analysis using the Microarray Platform Website) (Cited in the Office Action mailed 10 October 2024) in the Office Action mailed 06 June 2025 is withdrawn in view of the amendments received 06 October 2025.
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 pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 3, 26, and 28-30 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo et al. (Patent Pub: US 2004/0203037, Patent Pub Date: 14 October 2004) in view of Dhallen et al. (US Patent No.: 2004/0137470, Patent Pub Date 15 July 2004) in view of Kurn et al. (US2011/0189679 Pub: 04 August 2011, Cited in the Office Action mailed 06 June 2025) in view of Gong et al. (Cited in the Office Action mailed 10 October 2024) (PloS one, 2011-11, Vol.6 (11), p.e27156-e27156) in view of Li et al. (Journal of clinical oncology, 2006-04, Vol.24 (11), p.1754-1760).
Claim 3 step (a) recites isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, wherein the cfRNA is messenger RNA, and wherein isolating the cfRNA comprises adding a cell-membrane stabilizing agent to the test blood sample. Claim 3 step (b) recites reverse transcribing the cfRNA to obtain complementary DNA (cDNA). Claim 3 step (c) recites amplifying the cDNA to product double stranded cDNA amplification products. Claim 3 step (d) recites performing sequencing assay on the double stranded cDNA a0mplification products to obtain collection of sequencing data. Claim 3 step (d) recites the collection of sequence data comprises test fractional contributions of tissue types. Claim 3 step (e) recites computer processing the collection of sequencing data, wherein the computer processing comprises deconvolving the test fractional contributions of tissue types from the plasma derived from the test blood sample of the test subject against a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject. Claim 3 step (e) recites wherein deconvolving the test fractional contributions of tissue types further comprises (1) identifying a panel of tissue-associated transcripts, (2) determining total ribonucleic acid (RNA) in plasma derived from the test blood sample of the test subject, and (3) assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1), wherein the total RNA is considered a summation of the tissue-associated transcripts.
Lo et al. (Lo) discloses a method for diagnosing, monitoring, and predicting preeclampsia in pregnant women by determining the amount one or more mRNA species (i.e., total mRNA) in the pregnant women’s blood sample [claim 1 step (i)] and comparing the amount of mRNA (i.e., total mRNA) of step (i) to a standard control [par. 0044 and Lo claim 1]. Lo discloses the pregnant women’s sample as acellular [Lo, claim 7], as in instant claim 3 step (a) isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, wherein the cfRNA is messenger RNA (mRNA).
Lo discloses prior to the amplification step, a DNA (cDNA) copy of the mRNA of interest must be synthesized by reverse transcription [disclosure 0057], as in claim 3 step (b).
Lo discloses teaches quantitatively determining the amount of one or more mRNA species in the pregnant women blood such as those encoding hCRH, GAPDH [par. 0043, Lo claim 1 step (i) and claim 7], as in instant claim 3 step (e) sub step (1).
Lo discloses determining the amount of mRNA species (i.e., hCRH, GAPDH) [Lo, claim 1 step (i)], as in instant claim 3 step (e) sub step (2) determining total ribonucleic acid (RNA) in plasma derived from the test blood sample of the test subject. Here, determining the amount of mRNA species (i.e., hCRH, GAPDH) reads on determining the total amount of RNA because claim 1 of Lo teaches determining the “amount of mRNA species (i.e., hCRH, GAPDH) in the pregnant woman’s blood” which is the total amount of that mRNA species in the pregnant woman’s blood.
Dependent claim 30
Lo teaches “Prior to the amplification step, a DNA copy (cDNA) of the mRNA of interest must be synthesized. This is achieved by reverse transcription, which can be carried out as a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR), a modification of the polymerase chain reaction for amplifying RNA.” [disclosure page 5 right col para. 0057], as in instant claim 30. Here, the synthesize cDNA copies can be reverse transcribed, in a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR) which reads on performing PCR on cDNA libraries.
Lo does not teach wherein isolating the cfRNA comprises adding a cell-membrane stabilizing agent to the test blood sample claim 3 step (a). Lo does not explicitly teach amplifying the cDNA to product double stranded cDNA amplification products of claim 3 step (c). Lo does not teach performing sequencing assay on the double stranded cDNA amplification products to obtain collection of sequencing data of claim 3 step (d). Lo does not teach the collection of sequence data comprises test fractional contributions of tissue types of claim 3 step (d). Lo does not teach computer processing the collection of sequencing data, wherein the computer processing comprises deconvolving the test fractional contributions of tissue types from the plasma derived from the test blood sample of the test subject against a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject of claim 3 step (e). Lo does not teach wherein deconvolving the test fractional contributions of tissue types further comprises of claim 3 step (e). Lo does not teach assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1) of claim 3 step (e) sub step (3). Lo does not teach wherein the total RNA is considered a summation of the tissue-associated transcripts of claim 3 step (e) sub step (3). Lo does not teach claims 26 and 28-29.
Dhallan et al. (Dhallan) discloses that agents were added to sample to inhibit cell lysis such as membrane stabilizers, a cross-linker, or a cell lysis inhibitor [disclosure page 5 right col. para. 0057]. Dhallen discloses “membrane stabilizers agents such as may be added to the Sample including but not limited to aldehydes, urea formaldehyde, phenol formaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, Vitamin E, and Vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, Sucrose, astaxanthin, glucose, amitriptyline, isomer A hopane tetral phenylacetate, isomer Bhopane tetral phenylacetate, citicoline,inositol, Vitamin B, Vitamin B complex, cholesterol, hemisuccinate, Sorbitol, calcium, coenzyme Q, ubiquinone, Vitamin K, Vitamin K complex, menaquinone, zonegran, Zinc, ginkgo biloba extract, diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine, tegretol, PABA, disodium cromglycate, nedocromil Sodium, phenyloin, Zinc citrate, mexitil, dilantin, Sodium hyaluronate, or polaxamer 188.” [disclosure page 9 left col para. 0078] [Instant specification page 11 first paragraph]. Dhallan discloses “DNA that has been reverse transcribed from an RNA sample, such as cDNA. The Sequence of RNA can be determined according to the invention if it is capable of being made into a double stranded DNA form to be used as template DNA” [disclosure page 18 right col para. 0189], as in instant claim 3 wherein isolating the cfRNA comprises adding a cell-membrane stabilizing agent to the test blood sample.
It would be obvious to one of ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallan because Dhallen discloses methods analyzing nucleic acids (i.e., RNA) from cells. One of ordinary skill in the art would be motivated to combine Lo in view Dhallan because Dhallan discloses methods for using cell-membrane inhibitors and/or cell lysis inhibitors for quantifying free fetal DNA and discloses specific cell-membrane inhibitor agents (i.e., cell membrane stabilizers).Thus, there is a reasonable expectation of success using an acellular sample from a pregnant women of Lo and adding the cell lysis inhibitor of Dhallan to said acellular sample would a construct a claimed step that can isolate RNA from other cells or cells not previously removed in a sample and/or prevent intracellular mRNA contamination in order to yield a purified total RNA (i.e., mRNA) that can be utilized for deconvolving test fractional contributions of tissue types from a blood sample.
Kurn et al. (Kurn) discloses RNA can comprise cell-free RNA [claims 15, 32, and 48]. Kurn teaches reverse transcribed cDNA products [claim 18 and 34], as in instant claim 3 step (b).
Kurn discloses amplifying double stranded cDNA [claim 18 and 34], as in instant claim 3 step (c).
Kurn discloses sequencing the reversed transcribed products [claim 4-5], as in instant claim 3 step (d) performing sequencing assay on the double stranded cDNA amplification products to obtain collection of sequencing data.
Dependent claims 26 and 29
Kurn discloses using amplified cDNA can be from massively parallel sequencing enable by next generation sequencing technologies and platform, as representing by the RNA-seq data using Illumina’s Genome Analyzer [disclosure paragraph [0097]]. Kurn discloses “In one aspect, the invention provides for a method for whole transcriptome sequencing comprising providing a RNA sample, reverse transcribing the sample, amplifying the RNA sample using one or more primers to produce amplified products, and performing sequencing on the products [disclosure page 1 left col para. 0004], as in claim 26.
Kurn discloses generating double stranded cDNA product [claim 18 and 34], as in instant claim 29.
It would have been obvious to one of ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallan in view of Kurn because Kurn discloses methods for compositions and whole transcriptome analysis [title]. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallan in view of Kurn because Kurn discloses steps for generating double-stranded cDNA, and amplifying said double-stranded cDNA which can be utilized to process the mRNA species of Lo to determine total RNA species of a particular gene. Thus, there is a reasonable expectation of success to combine Lo in view of Dhallan in view of Kurn to produce RNA/mRNA data (i.e., total RNA) that can be subsequently summed and utilized for deconvolving test fractional contribution of tissues type and/or tissue types from a blood sample because the combination of Lo in view of Dhallan in view of Kurn which would yield a predictable method for generating double stranded cDNA via reverse transcription of cell-free RNA to produce test fractional contributions from the cell-free RNA data which could be further deconvoluted for determining tissue and/or cell type.
Gong also teaches deconvolving transcriptional profiling data [abstract]. Gong teaches deconvolution of transcriptional profiling data using quadratic programming with application to complex clinical blood samples [abstract]. Gong teaches method to various existing platforms to estimate proportions of different pure cell or tissue types and gene expression profiling of distinct phenotypes, with a focus on complex samples collected in clinical trials [abstract]. Gong teaches tissue types [page 2 table 1]. Gong teaches blood is a complex tissue type [page 3 right col deconvolution of circulating cells from whole blood sample second para.], as in claim 3 step (e) wherein the computer processing comprises deconvolving the test fractional contributions of tissue types.
Dependent claim 28
Gong also teaches deconvolving transcriptional profiling data [abstract]. Gong teaches deconvolution of transcriptional profiling data using quadratic programming with application to complex clinical blood samples [abstract], as in claim 28.
It would be obvious to one of ordinary skill in the art by the effective filing date of the claim invention to modify Lo in view of Dhallan in view of Kurn in view of Gong because Gong teaches methods for deconvolution of cell types. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallan in view of Kurn in view of Gong because Gong teaches methods for deconvolving cell and tissues type from RNA data but teaches deconvolving using quadratic programming for deconvolving RNA data for estimating proportions of different pure cell or tissue types and gene expression profiling of distinct phenotypes mixing fractions for more than ten species of circulating cells and to provide accurate estimates for relatively rare cell types [abstract]. Here, the would be a reasonable expectation of success combining Lo in view of Dhallan in view of Kurn in view of Gong because Gong teaches using quadratic programming for deconvolution of cell and tissue types. As such, combining Lo in view of Dhallan in view of Kurn in view of Gong would yield a predictable method using quadratic programming for deconvolution of tissue types.
Li teaches using a HGU133A microarrays for profiling and identifying the differences in serum mRNA transcriptomes between cancer patients and healthy controls. Li teaches analyzing 14,268 genes with the microarray [page 1756 right col second para.]. Li teaches using 35 control subjects [page 1756 left col results] as in claim 3 step (e) test blood sample of the test subject against a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject. Here, using the microarray teaches a reference cell-free transcriptome because the microarray, HGU133A, contains mRNA transcripts (i.e., 14,268 genes) for profiling and identifying transcript differences in serum mRNA transcriptomes between cancer and healthy patients. Furthermore, the use of healthy controls teaches the samples included reference sample (i.e., controls).
Li teaches “Ten significant upregulated candidates were selected from the list of 62 based on their reported cancer association: H3F3A, TPTl, FTHl, NCOA4, ARCR, THSMB, PRKCBl, FTLl, COX4Il, and SERPl (i.e., panel of identified tissue-associated transcripts)” which also teaches identifying a panel of tissue-associated transcripts of claim 3 step (e) sub step (1). Li teaches “five transcripts (H3F3A, TPTl , FTHl, NCOA4, and ARCR) were confirmed to be significantly elevated in OSCC sera” [page 1756 right col third para] which also teaches identifying a panel of tissue-associated transcripts of claim 3 step (e) sub step (1).
Li teaches regular qPCR was performed to quantify a subset of differently expressed transcripts in serum of OSCC patients (n = 32) versus controls (n = 35). Li teaches “regular PCR was performed on total RNA to obtain specific amplicons of the aforementioned subset of genes/transcripts. These amplicons underwent a serial dilution with the starting amount determined by a spectrophotometer. Those diluted amplicons, also used as positive controls, were run in parallel with the samples under identical qPCR conditions and amplified with the same set of primers.” [page 1756 Quantitative PCR], which also teaches determining a total RNA of claim 3 step (e) sub step (2).
Li et al. (Li) teach detecting oral squamous cell carcinoma (OSCC) in blood samples [abstract]. Li teaches serum circulating human mRNA profiling and its utility for oral cancer detection [abstract]. Li teaches microarrays were used to profile and identify the differences in serum mRNA transcriptomes between cancer and healthy patients [page 1756 right col second paragraph]. Li teaches “we identified 62 transcripts from the differently expressed 335 genes such that these 62 genes/transcripts (i.e., summed total mRNA) [Spec page 8 top of page] are all upregulated in OSCC serum.” [page 1756 right col second para]. Li teaches “Ten significant upregulated candidates were selected from the list of 62 based on their reported cancer association: H3F3A, TPTl, FTHl, NCOA4, ARCR, THSMB, PRKCBl, FTLl, COX4Il, and SERPl (i.e., panel of identified tissue-associated transcripts)” which also teaches identifying a panel of tissue-associated transcripts of claim 3 step (e) sub step (1). Li teaches “five transcripts (H3F3A, TPTl , FTHl, NCOA4, and ARCR) were confirmed to be significantly elevated in OSCC sera” [page 1756 right col third para], as in claim 3 step (e) sub step (3) assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1) and the total RNA is considered a summation of the tissue-associated transcripts of claim 3 step (e) sub step (3).
Here, Li teaches that the obtained amplicons of subset of genes/transcripts were run in parallel with the samples under identical qPCR conditions and amplified with the same set of primers which makes obvious that the total RNA (i.e., summed total mRNA transcripts) was assessed against the panel of tissue-associated transcripts because the total RNA and samples were run in parallel. Moreover, Li al teaches assessing a set of 62 transcripts (i.e., total summed mRNA of tissue-associated transcripts) against a set of 10 candidate transcripts (i.e., panel of tissue-associated transcripts). Therefore, it is obvious that Li teaches a method for deconvolving of tissue types.
It would be obvious to one of ordinary skill in the art by the effective filing date of the claim invention to modify Lo in view of Dhallan in view of Kurn in view of Gong in view of Li because Li teaches methods for analyzing serum circulating human mRNA detecting OSCC [abstract] and identifying mRNA transcripts associated with OSCC [page 1756 right col third para]. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallan in view of Kurn in view of Gong in view of Li because Li teaches methods using microarrays to profile and identify differences in serum mRNA transcriptomes, revealing five mRNA transcripts upregulated in OSCC, and using qPCR to quantify the transcripts. Here, there is a reasonable expectation of success combining Lo in view of Dhallan in view of Kurn in view of Gong in view of Li because the reference cell-free transcriptome in plasma, the identified mRNA transcripts data, the assessing of total RNA and panel of tissue-associated transcripts of Li could be incorporated into the methods of Lo, Dhallan, Kurn, and Gong for deconvolving tissue types. As such, combining Lo in view of Dhallan in view of Kurn in view of Gong in view of Li would yield a predictable method using a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject and assessing total RNA against a panel of tissue-associated transcripts for deconvolving tissue type of a sample(s).
Claim 12-13 and 16 is rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Gong in view of Li, as applied to claims 3, 26, and 28-30, and in further view of McGill (Tutorial: RMA Analysis using the Microarray Platform Website) (Cited in the Office Action mailed 06 June 2025).
Lo in view of Dhallen in view of Kurn in view of Gong in view of Li do not teach claims 12-13 and 16.
With respect to claim instant claim 12, the claim is rendered obvious because McGill teaches calcualting signal intensity of probes [page 3 Affymetrix arrays]. McGill teaches using RMA which that uses intensity values [page 4 RMA]. Lo discloses that oligonucleotide probes specific to mRNA binding (i.e., hCRH, hPL, KISS1, GAPDH) can be used to detect the presence of mRNA species and indicate the amount of mRNA species in comparison to the standard control, based on the intensity of the signal imparted by the probe [disclosure page 5 right col para. 0062]. Kurn discloses sequencing [Kurn, claims 4-5].
McGill et al. (McGill) teaches RMA analysis using microarray platform [title]. McGill teaches using Affymetrix GeneChip and calculating probe intensity for the expression of a targeted gene [page 3]. McGill teaches RMA properties that has probe intensities that are in log (base 2) scale [page 4], as in claim 13.
With respect to claim 16, the claim is rendered obvious because McGill teaches calcualting signal intensity of probes [page 3 Affymetrix arrays]. McGill teaches using RMA which that uses intensity values [page 4 RMA]. Kurn discloses using RNA-seq and quantification PCR [disclosure page 9 para 0093 example 3].
It would be obvious to one ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallen in view of Kurn in view of Gong in view of Li and in further view of McGill because McGill teaches analyzing probe intensities of microarray data using raw microarray algorithm (RMA). One of ordinary ski