The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This office action is in response to Applicants’ amendments/remarks received December 12, 2025.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claims 2-90, 94, 97, 99-100 are canceled. Claim 1, 91-93, 95-96, 98, 101-107, 108-116 are under consideration.
Priority: This application is a CIP of PCT/US2019/058961, filed October 30, 2019, which claims benefit of provisional application 62/752533, filed October 30, 2018.
Objections and Rejections
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 91-93, 95-96, 98, 101-107, 108-116 are rejected under 35 U.S.C. 103 as being unpatentable over Nerenberg et al. (WO 2019060369; IDS 06.13.23, previously cited) in view of Kukurba et al. (Cold Spring Harb Protoc. 2015(11): 951-69. doi: 10.1101/pdb.top084970; previously cited), Mercer et al. (2014 Nature Protocols 9(5): 989-1009; previously cited), and Illumina (Illumina TruSeq® RNA sample preparation Guide 2014: 132 pages).
Nerenberg et al. disclose methods and systems for detecting tissue conditions comprising quantifying and analyzing cell free polynucleotides, comparing levels of the polynucleotides to a reference (at least abstract, p. 1-8). Nerenberg et al. disclose a method for isolating cell free ribonucleic acid (RNA) sample, the method comprising centrifuging blood samples in EDTA tubes at 1900g to obtain a plasma sample (p. 28). Nerenberg et al. disclose centrifuging the plasma at 16000g and passing through a 0.8 µm filter (p. 28); purifying or isolating RNA from the lipidic and proteinaceous structures by incubating the sample with chaotropic salts, proteinase, Tris, etc. to disrupt the lipidic and proteinaceous structures; passing the sample through a silica membrane; eluting the nucleic acids with low ionic buffers or water; treating the samples containing cfRNA with DNase to eliminate DNA; quantifying the cfRNA (p. 28; instant claims 1(a) to 1(c), 98, 114).
Since Nerenberg et al. disclose a method comprising the same steps and features recited in at least instant claims 1(a) to 1(b), including centrifuging a cell-free biological sample at a speed 16,000g and isolating the cfRNA with a silica membrane, it would follow that the centrifuging and isolating (or enriching) steps in Nerenberg et al. result in separating an extracellular vesicle, including exosome, from the plasma sample and therefore, the purifying and/or isolating RNA from the lipidic and proteinaceous structures disclosed in Nerenberg et al. also purifies RNA from the extracellular vesicle and as a result, comprises the noted components and non-blood genes in instant claims 1, 98, 114.
Nerenberg et al. further disclose that the methods also provide for quantifying RNA, or a cDNA thereof, in a sample from a subject; some methods comprise amplifying the RNA, or cDNA, thereof, where the amplifying comprises hybridizing the RNA or cDNA with a random hexamer (at least paragraph 0075, p. 25). Nerenberg et al. disclose in some instances, the nucleic acid is cDNA; in some instances, the reagent is an oligonucleotide that is complementary to a nucleic acid of interest; in some instances, the reagent is a reverse transcriptase, and the system is used to reverse transcribe the RNA to a cDNA; in some instances, the reagent is an oligonucleotide that is complementary to the cDNA; in some instances, the system comprises two oligonucleotides capable of amplifying the RNA or cDNA; in some instances, the reagent is an enzyme that amplifies the nucleic acid; in some instances the systems comprise a machine or reagent necessary for producing and/or reading Q-PCR or sequencing readout; such machines and reagents are well known in the art (at least paragraph 0085); in some instances, the systems and methods described herein comprise methods to quantify relative amount of a specific cell type/tissue/organ of interest from a complex mixture, such as cell-free RNA, based gene expression measurements of the mixture (at least paragraph 0086). Nerenberg et al. do not explicitly teach sequencing the RNA and preparing a cDNA library.
Kukurba et al. disclose RNA molecules are essential for interpreting the functional elements of the genome and understanding development and disease (at least p. 951). Kukurba et al. disclose RNA-sequencing (RNA-Seq) uses the high-throughput sequencing methods to provide insight into the transcriptome of a cell (at least p. 951). Kukurba et al. disclose a typical RNA-Seq experiment involves extracting RNA from the biological material of choice (e.g. cells, tissues); isolating the RNA molecules; converting the RNA to cDNA by reverse transcription; fragmenting or amplifying randomly primed cDNA molecules; preparing or constructing the sequencing library; sequencing it on an NGS platform (at least p. 952-953, also Fig. 1). Therefore, Kukurba et al. also disclose utilizing random oligos to prime the RNA to generate the cDNA library (p. 954). Kukurba et al. disclose approaches, including PCR-based approaches, hybrid capture, in-solution capture, molecular inversion probes, have been developed to selectively enrich regions of interest (at least p. 955).
Mercer et al. disclose targeted RNAseq can focus on sequencing targeted genes of interest (at least p. 989). Mercer et al. disclose labeled in-solution DNA oligonucleotides capture targets of interest that are then subjected to sequencing (at least p. 989). Mercer et al. disclose in-solution capture uses labeled RNA or DNA oligonucleotides that can be hybridized to cRNA or cDNA of interest (at least p. 990). Mercer et al. disclose the targeted RNAseq is used with Illumina TruSEQ RNA sample preparation (p. 997), where Illumina discloses that the RNA are reverse transcribed to cDNA using reverse transcriptase and random primers (p. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the RNA-Seq steps disclosed in Kukurba et al. and Mercer et al., which include performing reverse transcription of RNA to cDNA by contacting the RNA with random hexamers to prime the RNA, forming double-stranded cDNA, preparing a cDNA library, contacting the cDNA library with baits representative of the target region, and sequencing the cDNA library to produce sequencing reads, to the method for quantifying and identifying cell-free RNA of Nerenberg et al. noted above, and thereby arriving at the claimed method (instant claims 1(a) to 1(h), 98, 114). The motivation to do so is given by the prior art which disclose RNA-seq is a known laboratory technique for quantifying and identifying gene expression in cells and tissues of interest. One of ordinary skill would have a reasonable expectation of success because methods for extracting and isolating RNA and RNA-sequencing are known and disclosed in the prior art.
Regarding instant claim 91, Nerenberg et al. disclose the sample is a blood plasma sample (at least paragraph 0033, also p. 28-29).
Regarding instant claims 92-93, 95, Nerenberg et al. disclose centrifuging the sample more than once (at least paragraph 0057), including centrifuging a blood sample at 1900g for 10 min.; then centrifuging the plasma sample at 16000g and passing through a 0.8 µm filter (p. 28); and then purifying or isolating RNA from the lipidic and proteinaceous structures (p. 28).
Regarding instant claim 96, Nerenberg et al. disclose centrifuging the sample at 5000g to 25000g, including 12000g to 18000g (at least paragraph 0057).
Regarding instant claim 101, Nerenberg et al. disclose the cell-depleted sample is contacted with a silica column; incubating the cell-depleted sample with a chaotropic salt to enhance binding of the RNA to the silica column or silica membrane (at least paragraph 0003, p. 28-29). Therefore, it would be obvious to one of ordinary skill to purify the cell-depleted RNA by contacting the RNA sample with a silica column more than one time, where the silica column functions as an affinity column, desalting column, and has a silica membrane.
Regarding instant claims 102-103, as noted above, Nerenberg et al. disclose a method comprising the same steps and features recited in at least instant claims 1(a) to 1(b), including centrifuging a cell-free biological sample at a speed 16,000g and isolating/purifying the cfRNA comprising binding RNA to a silica column and passing through a silica membrane to separate the RNA from lipidic and proteinaceous structures (p. 28). Therefore, it would follow that the method of Nerenberg et al results in depletion of ribosomal RNA from the isolated RNA and therefore enriches for the isolated RNA, which is a protein-coding nucleotide sequence.
Regarding instant claim 104, Nerenberg et al. disclose the RNA is from tissue including liver, neurological tissue, kidney, etc. (at least paragraph 0071).
Regarding instant claims 105-106, Mercer et al. disclose preparing an indexed cDNA library with a unique index and sequencing the indexed cDNA library (at least p. 994, 998-1000) and Kukurba et al. also disclose sequencing indexed libraries (at least p. 956).
Regarding instant claim 107, Kukurba et al. disclose aligning the sequence reads to a reference genome (at least p. 959, 960) and Mercer et al. also disclose aligning the reads to a reference genome (at least p. 1007).
Regarding instant claim 108, Illumina discloses adding concentrations of random hexamers for RT priming (at least p. 20-21, 60). It is known that “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Therefore, it would have been obvious to one of ordinary skill to arrive at the recited concentration of at least 200 micromolar random oligonucleotides by routine optimization.
Regarding instant claim 109, Mercer et al. disclose that alternative in-solution oligonucleotide capture that are available in the prior art include exome enrichment (p. 990). Therefore, it would be obvious that the baits can comprise whole exome baits.
Regarding instant claim 110, Mercer et al. also disclose adding RNA spike-in mixes to the RNA sample before capture (at least p. 993-994, 997). Therefore, it would be obvious that the purified or isolated RNA samples can be spiked with external reference RNAs.
Regarding instant claim 111, Nerenberg et al. disclose purifying or isolating RNA from the lipidic and proteinaceous structures by incubating the sample at 60C with chaotropic salts, proteinase, Tris, etc. to disrupt the lipidic and proteinaceous structures (at least p. 28). Therefore, it would be obvious to contact the cell-free samples with a pre-warmed lysis buffer.
Regarding instant claim 112, Nerenberg et al. disclose subjecting the RNA samples through silica membrane with washing for at least two times (at least p. 28-29).
Regarding instant claim 113, Nerenberg et al. disclose eluting the nucleic acids from the silica membrane (at least p. 28-29). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Therefore, it would have been obvious to one of ordinary skill to arrive at a plurality of elution steps for the RNA from the silica membrane by routine optimization.
Regarding instant claim 114, since Nerenberg et al. disclose a method comprising the same steps and features recited in at least instant claims 1(a) to 1(b), including centrifuging a cell-free biological sample at a speed 16,000g and isolating the cfRNA with a silica membrane, it would follow that the centrifuging and isolating (or enriching) steps in Nerenberg et al. result in separating an extracellular vesicle, including exosome, from the plasma sample and therefore, the purifying and/or isolating RNA from the lipidic and proteinaceous structures disclosed in Nerenberg et al. also purifies RNA from the extracellular vesicle and as a result, comprises the noted components and non-blood genes in instant claims 1, 98, 114.
Regarding instant claims 115-116, Nerenberg et al. disclose the amplifying comprises hybridizing the RNA or cDNA with a random hexamer (at least paragraph 0075, p. 25), Illumina discloses that the RNA are reverse transcribed to cDNA using reverse transcriptase and random hexamers (p. 20, 21), and Mercer et al. disclose increased efficiency of targeted RNASeq reduces reagent costs (at least p. 989).
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive.
Applicants assert that instant claim 1, part (d), has been amended to recite performing the reverse transcription with random oligonucleotides configured to prime the isolated RNA. Applicants assert that the cited references do not teach or disclose (c) contacting the isolated RNA with a deoxyribonuclease and (d) performing reverse transcription on the RNA with random oligonucleotides configured to prime the isolated RNA.
Applicants’ remarks are not persuasive. As noted in the 103 rejection, to isolate the cell free RNA, Nerenberg et al. disclose centrifuging the plasma at 16000g and passing through a 0.8 µm filter (p. 28); purifying or isolating RNA from the lipidic and proteinaceous structures by incubating the sample with chaotropic salts, proteinase, Tris, etc. to disrupt the lipidic and proteinaceous structures; passing the sample through a silica membrane; eluting the nucleic acids with low ionic buffers or water; treating the samples containing cfRNA with DNase to eliminate DNA; quantifying the cfRNA (p. 28; instant claims 1(a) to 1(c), 98, 114). Therefore, Nerenberg et a. fairly disclose instant claim 1(c), contacting the isolated RNA with DNase (or deoxyribonuclease).
Regarding instant claim 1(d), as noted above, Nerenberg et al. disclose quantifying the cfRNA (p. 28). Nerenberg et al. disclose that the methods for quantifying RNA, or a cDNA comprise amplifying the RNA, or cDNA, thereof, where the amplifying comprises hybridizing the RNA or cDNA with a random hexamer (at least paragraph 0075, p. 25). Therefore, Nerenberg et al. fairly disclose priming the isolated RNA with random hexamers or oligonucleotides.
Nerenberg et al. disclose that the systems and methods described comprise methods to quantify relative amount of a specific cell type/tissue/organ of interest from a complex mixture, such as cell-free RNA, based gene expression measurements of the mixture (at least paragraph 0086).
Nerenberg et al. is also further cited with at least Kukurba et al., which disclose utilizing random oligos to prime the RNA to generate the cDNA library (Kukurba et al. p. 954) and Mercer et al., which disclose utilizing targeted RNAseq with Illumina TruSEQ RNA sample preparation (Mercer et al. p. 997), where Illumina discloses that the RNA are reverse transcribed to cDNA using reverse transcriptase and random primers (Illumina p. 2).
Therefore, the steps recited in instant claim 1 for preparing a cell-free RNA sample, including the recited steps of 1(c) contacting the isolated RNA with deoxyribonuclease and 1(d) performing reverse transcriptase on the isolated RNA with random hexamers are all well-known and recognized steps for preparing RNA samples for RNA sequencing and analysis.
Regarding Applicants’ remarks that the instant specification discloses that implementation of random hexamers improve RNA to cDNA conversion efficiency and quantification accuracy from cfRNA, the remarks are not persuasive. Mercer et al. disclose increased efficiency of utilizing targeted RNASeq (p. 989), which therefore, also includes performing reverse transcriptase on the isolated RNA with random hexamers (at least p. 996-998). Therefore, Applicants’ remarks regarding increased efficiency with implementation of random hexamers are not found persuasive.
For at least these reasons, the claims remain rejected under 35 U.S.C. 103.
Claims 1, 91-93, 95-96, 98, 101-107, 108-116 are rejected under 35 U.S.C. 103 as being unpatentable over Koh et al. (2014 PNAS 111(20): 7361-7366; IDS 06.13.23, previously cited), Qiagen (Qiagen RNeasy Micro Handbook 2014: 84 pages) in view of Kukurba et al. (Cold Spring Harb Protoc. 2015(11): 951-69. doi: 10.1101/pdb.top084970), Mercer et al. (2014 Nature Protocols 9(5): 989-1009) and Illumina (Illumina TruSeq® RNA sample preparation Guide 2014: 132 pages). Koh et al. disclose circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs (at least p. 7361). Koh et al. disclose a method for isolating a RNA sample, the method comprising centrifuging blood samples in EDTA tubes at 1600g for 10 mins., and then centrifuging the obtained plasma samples at 16,000 g for 10 mins., and then isolating the RNA from the plasma sample (p. 7364; instant claims 1, 91, 98). Koh et al. disclose the cell-free RNA is extracted from plasma and purified by Qiagen RNeasy kit with DNase I digestion (at least p. 7364; instant claims 1, 98, 114), where it is disclosed that the Qiagen RNeasy technology combines the selective binding properties of a silica-based membrane with the speed of microspin technology (Qiagen at least p. 7).
Since Koh et al. disclose a method comprising the same steps and features recited in at least instant claims 1(a) to 1(c), including centrifuging a cell-free biological sample at a speed 16,000g and isolating the cfRNA with a silica membrane, it would follow that the centrifuging and isolating (or enriching) steps in Koh et al. result in separating an extracellular vesicle, including exosome, from the plasma sample and therefore, the purifying and/or isolating RNA from the lipidic and proteinaceous structures disclosed in Koh et al. also purifies RNA from the extracellular vesicle and as a result, comprises the noted non-blood genes in instant claims 1, 98, 114.
Koh et al. further disclose cDNA synthesis and amplification from the cell-free RNA using an RNA-Seq system kit, followed by sequencing to produce sequence reads (at least p. 7361-7362, 7364). Koh et al. do not explicitly teach the steps of sequencing the RNA and preparing a cDNA library.
Kukurba et al. disclose RNA molecules are essential for interpreting the functional elements of the genome and understanding development and disease (at least p. 951). Kukurba et al. disclose RNA-sequencing (RNA-Seq) uses the high-throughput sequencing methods to provide insight into the transcriptome of a cell (at least p. 951). Kukurba et al. disclose a typical RNA-Seq experiment involves extracting RNA from the biological material of choice (e.g. cells, tissues); isolating the RNA molecules; converting the RNA to cDNA by reverse transcription; fragmenting or amplifying randomly primed cDNA molecules; preparing or constructing the sequencing library; sequencing it on an NGS platform (at least p. 952-953, also Fig. 1). Therefore, Kukurba et al. also disclose utilizing random oligos to prime the RNA to generate the cDNA library (p. 954). Kukurba et al. disclose approaches, including PCR-based approaches, hybrid capture, in-solution capture, molecular inversion probes, have been developed to selectively enrich regions of interest (at least p. 955).
Mercer et al. disclose targeted RNAseq can focus on sequencing targeted genes of interest (at least p. 989). Mercer et al. disclose labeled in-solution DNA oligonucleotides capture targets of interest that are then subjected to sequencing (at least p. 989). Mercer et al. disclose in-solution capture uses labeled RNA or DNA oligonucleotides that can be hybridized to cRNA or cDNA of interest (at least p. 990). Mercer et al. disclose the targeted RNAseq is used with Illumina TruSEQ RNA sample preparation (p. 997), where Illumina discloses that the RNA are reverse transcribed to cDNA using reverse transcriptase and random primers (p. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the RNA-Seq steps disclosed in Kukurba et al. and Mercer et al., which include performing reverse transcription of RNA to cDNA by contacting the RNA with random hexamers to prime the RNA, forming double-stranded cDNA, preparing a cDNA library, contacting the cDNA library with baits representative of the target region, and sequencing the cDNA library to produce sequencing reads, to the method for quantifying and identifying cell-free RNA of Koh et al. noted above, and thereby arriving at the claimed method (instant claims 1(a) to 1(h), 98, 114). The motivation to do so is given by the prior art which disclose RNA-seq is a known laboratory technique for quantifying and identifying gene expression in cells and tissues of interest. One of ordinary skill would have a reasonable expectation of success because methods for extracting and isolating RNA and RNA-sequencing are known and disclosed in the prior art.
Regarding instant claim 91, Koh et al. disclose the sample is a blood plasma sample (at least p. 7364).
Regarding instant claims 92-93, Koh et al. disclose centrifuging the plasma at 16000g for 10 mins. (p. 7364); and then purifying or isolating RNA (p. 7364).
Regarding instant claim 95, Koh et al. disclose the cell-free RNA is extracted from plasma and purified by Qiagen RNeasy kit with DNase I digestion (at least p. 7364), where it is disclosed that the Qiagen RNeasy procedure enriches for mRNA (RNA molecules >200 nucleotides) by the RNeasy MinElute spin column membrane (Qiagen p. 7, 13-14), where the RNeasy MinElute spin column membrane is utilized more than one time (at least p. 25-33). Therefore, it would be obvious to one of ordinary skill that the method of Koh et al. isolates and purifies cell-free RNA based on size exclusion and where the spin column membrane functions as a filter.
Regarding instant claim 96, Koh et al. disclose centrifuging the plasma at 16000g for 10 mins. (p. 7364); and then purifying or isolating RNA (p. 7364). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05. Therefore, it would have been obvious for one of ordinary skill to arrive at the recited centrifugation speeds 10000 to 15000g, including 12000g, by routine optimization and/or experimentation because the recited speeds are similar to what is disclosed in the prior art.
Regarding instant claim 101, as noted above, Koh et al. disclose purifying and/or isolating RNA using the RNeasy kit (Qiagen) according to the manufacturer’s instructions, where the Qiagen RNeasy kit utilizes silica technology including a silica membrane spin column, where the RNeasy MinElute spin column membrane is utilized more than one time (at least p. 25-33). Therefore, it would be obvious to one of ordinary skill to purify the cell-depleted RNA by contacting the RNA sample with a silica column more than one time, where the silica column functions as an affinity column, desalting column, and has a silica membrane.
Regarding instant claims 102-103, as noted above, Koh et al. disclose a method comprising the same features and steps recited in at least instant claims 1(a) to 1(b), including centrifuging a cell-free biological sample at a speed 16,000g, and further disclose purifying and/or isolating RNA using the RNeasy kit (Qiagen) according to the manufacturer’s instructions, which would include separating RNA from lipidic and proteinaceous structures (p. 7364). Therefore, it would follow that the method of Koh et al. results in depletion of ribosomal RNA from the isolated RNA and therefore enriches for the isolated RNA, which is a protein-coding nucleotide sequence.
Regarding instant claim 104, Koh et al. disclose RNA from different tissues types, including bone marrow, lymph nodes, smooth muscle, epithelial cells, etc. (at least p. 7362).
Regarding instant claims 105-106, Mercer et al. disclose preparing an indexed cDNA library with a unique index and sequencing the indexed cDNA library (at least p. 994, 998-1000) and Kukurba et al. also disclose sequencing indexed libraries (at least p. 956).
Regarding instant claim 107, Koh et al. disclose mapping the sequence reads against the human reference genome (at least p. 7362), Kukurba et al. disclose aligning the sequence reads to a reference genome (at least p. 959, 960), and Mercer et al. also disclose aligning the reads to a reference genome (at least p. 1007).
Regarding instant claim 108, Illumina discloses adding concentrations of random hexamers for RT priming (at least p. 20-21, 60). It is known that “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Therefore, it would have been obvious to one of ordinary skill to arrive at the recited concentration of at least 200 micromolar random oligonucleotides by routine optimization.
Regarding instant claim 109, Mercer et al. disclose that alternative in-solution oligonucleotide capture that are available in the prior art include exome enrichment (p. 990). Therefore, it would be obvious that the baits can comprise whole exome baits.
Regarding instant claim 110, Mercer et al. also disclose adding RNA spike-in mixes to the RNA sample before capture (at least p. 993-994, 997). Therefore, it would be obvious that the purified or isolated RNA samples can be spiked with external reference RNAs.
Regarding instant claim 111, Koh et al. disclose the cell-free RNA is extracted from plasma and purified by Qiagen RNeasy kit with DNase I digestion (at least p. 7364; instant claims 1, 98, 114), where it is disclosed that the Qiagen RNeasy technology also utilizes an RTL buffer (lysis buffer) (Qiagen at least p. 7, 13, 15), where buffer RTL may form a precipitate during storage and if necessary, is dissolved by warming (at least p. 24, 32, 41, 50, 55). Therefore, it would be obvious to contact the cell-free samples with a pre-warmed lysis buffer.
Regarding instant claim 112, as noted, Koh et al. disclose the cell-free RNA is extracted from plasma and purified by Qiagen RNeasy kit with DNase I digestion (at least p. 7364; instant claims 1, 98, 114), where it is disclosed that the Qiagen RNeasy technology combines the selective binding properties of a silica-based membrane with the speed of microspin technology (Qiagen at least p. 7). Qiagen discloses subjecting the RNA samples through silica-based membrane columns with washing for at least two times (at least p. 26-29).
Regarding instant claim 113, as noted, Koh et al. disclose the cell-free RNA is extracted from plasma and purified by Qiagen RNeasy kit with DNase I digestion (at least p. 7364; instant claims 1, 98, 114), where it is disclosed that the Qiagen RNeasy technology combines the selective binding properties of a silica-based membrane with the speed of microspin technology (Qiagen at least p. 7). Qiagen discloses eluting the RNA from the silica-based membrane columns (at least p. 28-29). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Therefore, it would have been obvious to one of ordinary skill to arrive at a plurality of elution steps for the RNA from the silica-based membrane column by routine optimization.
Regarding instant claim 114, since Koh et al. disclose a method comprising the same steps and features recited in at least instant claims 1(a) to 1(b), including centrifuging a cell-free biological sample at a speed 16,000g and isolating the cfRNA with a silica membrane, it would follow that the centrifuging and isolating (or enriching) steps in Koh et al. result in separating an extracellular vesicle, including exosome, from the plasma sample and therefore, the purifying and/or isolating RNA from the lipidic and proteinaceous structures disclosed in Koh et al. also purifies RNA from the extracellular vesicle and as a result, comprises the noted non-blood genes in instant claims 1, 98, 114.
Regarding instant claims 115-116, Illumina discloses that the RNA are reverse transcribed to cDNA using reverse transcriptase and random hexamers (p. 20, 21) and Mercer et al. disclose increased efficiency of targeted RNASeq reduces reagent costs (at least p. 989).
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive.
Applicants assert that instant claim 1, part (d), has been amended to recite performing the reverse transcription with random oligonucleotides configured to prime the isolated RNA. Applicants assert that the cited references do not teach or disclose (c) contacting the isolated RNA with a deoxyribonuclease and (d) performing reverse transcription on the RNA with random oligonucleotides configured to prime the isolated RNA.
Applicants’ remarks are not persuasive. As noted in the 103 rejection, to isolate the cell free RNA, Koh et al. disclose centrifuging blood samples in EDTA tubes at 1600g for 10 mins., and then centrifuging the obtained plasma samples at 16,000 g for 10 mins., and then isolating the RNA from the plasma sample (p. 7364; instant claims 1, 91, 98). Koh et al. disclose the cell-free RNA is extracted from plasma and purified by Qiagen RNeasy kit with DNase I digestion (at least p. 7364; instant claims 1, 98, 114), where it is disclosed that the Qiagen RNeasy technology combines the selective binding properties of a silica-based membrane with the speed of microspin technology (Qiagen at least p. 7). Therefore, Koh et a. fairly disclose instant claim 1(c), contacting the isolated RNA with DNase (or deoxyribonuclease).
Regarding instant claim 1(d), Koh et al. further disclose cDNA synthesis and amplification from the cell-free RNA using an RNA-Seq system kit, followed by sequencing to produce sequence reads (at least p. 7361-7362, 7364).
Koh et al. is further cited with at least Kukurba et al., which disclose utilizing random oligos to prime the RNA to generate the cDNA library (Kukurba et al. p. 954) and Mercer et al., which disclose utilizing targeted RNAseq with Illumina TruSEQ RNA sample preparation (Mercer et al. p. 997), where Illumina discloses that the RNA are reverse transcribed to cDNA using reverse transcriptase and random primers (Illumina p. 2).
Therefore, the steps recited in instant claim 1 for preparing a cell-free RNA sample, including the recited steps of 1(c) contacting the isolated RNA with deoxyribonuclease and 1(d) performing reverse transcriptase on the isolated RNA with random hexamers are all well-known and recognized steps for preparing RNA samples for RNA sequencing and analysis.
Regarding Applicants’ remarks that the instant specification discloses that implementation of random hexamers improve RNA to cDNA conversion efficiency and quantification accuracy from cfRNA, the remarks are not persuasive. Mercer et al. disclose increased efficiency of utilizing targeted RNASeq (p. 989), which therefore, also includes performing reverse transcriptase on the isolated RNA with random hexamers (at least p. 996-998). Therefore, Applicants’ remarks regarding increased efficiency with implementation of random hexamers are not found persuasive.
For at least these reasons, the claims remain rejected under 35 U.S.C. 103.
No claim is allowed.
THIS ACTION IS MADE FINAL. 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Marsha Tsay whose telephone number is (571)272-2938. The examiner can normally be reached M-F.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath N. Rao can be reached on 571-272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Marsha Tsay/Primary Examiner, Art Unit 1656