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 .
DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 11/12/2025 has been entered.
Application status
In response to the previous Office action, a Final rejection (mailed on 05/19/2025), Applicants filed a response and amendment received on 11/12/2025. Said amendment canceled Claims 9-10 and 28-61, amended Claims 1 and 20-21, and added Claims 64-68. Thus, Claims 1-8, 11-27 and 62-68 are at issue and present for examination.
Claim Rejections - 35 U.S.C. § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The previous rejection of Claim 1-8, 11-27 and 62-63 under 35 U.S.C. 103 as being unpatentable over Shum et al. (WO2018/144240, publication date 08/09/2018, see IDS) in view of Krjutskov et al. (Globin mRNA reduction for whole-blood transcriptome sequencing, Sci Rep, 2016 Aug 12:6:31584), QIAGEN OneStep RT-PCR handbook (retrieved from the internet: << https://www.qiagen.com/be/resources/download.aspx?id=57743726-84e1-423a-9d8f-a3fa89bbe7eb&lang=en>>, retrieved on 09/11/2024, which was published on October 2012), Becker et al. (US Patent No. 7713697) and Chim et al. (US Patent No. 10683557) is withdrawn in favor of a new grounds for rejection as shown below.
Claim 1-8, 11-27 and 62-68 are rejected under 35 U.S.C. 103 as being unpatentable over Vries et al. (A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples, Plos One, January 2011, Volume 6, Issue 1, e16118, pages 1-9) in view of Shum et al. (WO2018/144240, publication date 08/09/2018, see IDS), Krjutskov et al. (Globin mRNA reduction for whole-blood transcriptome sequencing, Sci Rep, 2016 Aug 12:6:31584), QIAGEN OneStep RT-PCR handbook (retrieved from the internet: << https://www.qiagen.com/be/resources/download.aspx?id=57743726-84e1-423a-9d8f-a3fa89bbe7eb&lang=en>>, retrieved on 09/11/2024, which was published on October 2012), Becker et al. (US Patent No. 7713697) and Chim et al. (US Patent No. 10683557).
The instant claims are drawn to a method for inhibiting cDNA synthesis of one or more unwanted RNA species in an RNA sample during reverse transcription, comprising: (a) providing an RNA sample that comprises one or more desired RNA species and one or more unwanted RNA species, (b) annealing one or more blocking oligonucleotides to one or more regions of the one or more unwanted RNA species in the RNA sample to generate a template mixture, wherein the one or more blocking oligonucleotides are complementary, and stably bind, to the one or more regions of the one or more unwanted RNA species, and comprise 3' modifications that prevent the one or more blocking oligonucleotides from being extended, wherein the number of the one or more blocking oligonucleotides is at least 10, and wherein two or more of the blocking oligonucleotides anneal to different regions of at least one of the one or more unwanted RNA species, and (c) incubating the template mixture with a reaction mixture that comprises: (i) at least one reverse transcriptase,(ii) one or more reverse transcription primers and (iii) a reaction buffer, under conditions sufficient to synthesize cDNA molecules using the one or more desired RNA species as template(s), wherein cDNA synthesis using the one or more unwanted RNA species is inhibited.
Vries et al. teach a method for inhibiting cDNA synthesis of one or more unwanted RNA species in an RNA sample during reverse transcription, comprising: (a) providing an RNA sample that comprises one or more desired RNA species and one or more unwanted RNA species, (b) annealing one or more blocking oligonucleotides (BO) to one or more regions of the one or more unwanted RNA species in the RNA sample to generate a template mixture, wherein the one or more blocking oligonucleotides are complementary, and stably bind, to the one or more regions of the one or more unwanted RNA species, and comprise 3' modifications that prevent the one or more blocking oligonucleotides from being extended, wherein the number of the one or more blocking oligonucleotides is at least 10, and wherein two or more of the blocking oligonucleotides anneal to different regions of at least one of the one or more unwanted RNA species, and (c) incubating the template mixture with a reaction mixture that comprises: (i) at least one reverse transcriptase, (ii) one or more reverse transcription primers and (iii) a reaction buffer, under conditions sufficient to synthesize cDNA molecules using the one or more desired RNA species as template(s), wherein cDNA synthesis using the one or more unwanted RNA species is inhibited (see section under “3) rRNA-blocking oligos in the reverse transcription reaction” on page 3, and VIDISCA on page 7).
Vries et al. specifically teach said method comprising the use of 10 µM rRNA-blocking oligonucleotides comprising 18S rRNA and 28S rRNA, which contained a 3’ dideoxy C6 amino modification as an LNA, which were:
(1) 1-Morrna 59 CTTTCGCTCTGGTCCGT 39 –C6 [18S, nt. 977–1071];
(2) 2-Morrna 59 CACTAATTAGATGACGAGG 39–C6 [28S, nt. 3767–3785];
(3) 3-Morrna 59 TGACATTCAGAGCACTGG 39–C6 [28S, nt. 3679–3696];
(4) 4-Morrna 59 GTTACTGAGGGAATCCTG 39 –C6 [28S, nt. 72–89]; and
(5) 5-Morrna 59 CACCAGTTCTAAGTCGG 39–C6 [28S, nt. 3580–3596],
wherein the distance between two neighboring regions is between 0-100, i.e., 3679 (3) – 3596 (5) = 83 nucleotides, thereby reading on claims 1-6, 8, 12-16, 21-22, 24-27 and 62-68 (see section under “3) rRNA-blocking oligos in the reverse transcription reaction” on page 3; and under VIDISCA on page 7).
Since the Office does not have the facilities for examining and comparing the number/amount of blocking oligonucleotides used by applicants', i.e., at least 10 or about 0.1 to 50 pmol per blocking oligonucleotide, with that of Vries et al., i.e., 10 µM rRNA-blocking oligonucleotides, the burden is on the applicant to show a novel or unobvious difference between the claimed amount and the number/amount used in the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594.
Vries et al. do not teach [1] the use of specific temperatures, i.e., at least 65, 70, or 75 degrees Celsius, subsequently reducing the temperature to lower than 40 degrees Celsius (claims 7 and 20); [2] the different regions are evenly distributed (claim 11); [3] the use of salt of KCl and its concentrations (claims 17-18); and [4] the RNA sample is from whole blood, serum or plasma.
Shum et al. teach a method of selective amplification, comprising: providing a sample comprising a plurality of nucleic acid target molecules and one or more undesirable nucleic acid species; providing a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence and a binding region; contacting the plurality of oligonucleotide probes with the plurality of nucleic acid target molecules for hybridization; extending oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules to generate a plurality of extension products using a reverse transcriptase and/or a polymerase (see claim 72); providing a blocking oligonucleotide that specifically binds to at least one of the one or more undesirable nucleic acid species; and amplifying the plurality of extension products to generate a plurality of amplicons,
whereby the amplification or the extension of the undesirable nucleic acid species is reduced by the blocking oligonucleotide (claim 1),
wherein the blocking oligonucleotide is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNAIDNA chimera, or a PNA1DNA chimera (see claim 3),
wherein the blocking oligonucleotide is unable to function as a primer for a reverse transcriptase or a polymerase (see claim 10),
wherein the blocking oligonucleotide is 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 200 nt long or any range thereof including being fully complementary (see para [0053] and claim 16),
wherein the blocking oligonucleotide has a Tm of at least 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C (see para [0051] and claim 9),
wherein said method comprising providing blocking oligonucleotides that specifically binds to at least 100 undesirable nucleic acid species in the sample (see claim 6), which meets the newly added limitation of “wherein the number of the one or more blocking oligonucleotides is at least 10” in Applicants’ claim 1, wherein the undesirable nucleic acid species comprises rRNA, mtRNA, genomic DNA, intronic sequence, high abundance sequence, and any combination thereof (see claim 23), wherein the plurality of nucleic acid target molecules comprises mRNA target molecules (see claim 51), wherein the plurality of amplicons comprises a cDNA library thereby constructing a sequencing library (see para [0072]).
Shum et al. teach that the plurality of blocking oligonucleotides can specifically bind to at least 1, at least 2, at least 5, at least 10, at least 100, at least 1,000 or more of the one or more undesirable nucleic acid species, and that in some embodiments, the blocking oligonucleotide can specifically bind to within 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, or 1,000 nt of the 5' end of at least one of the one or more undesirable nucleic acid species…, blocking oligonucleotide can specifically binds to a sequence close to the 3' end of the undesirable nucleic acid species, for example, the blocking oligonucleotide can specifically bind to within 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, l 00 nt, 200 nt, 300 nt, 400 nt, 500 nt, 1,000 nt of the 3' end of at least one of the one or more undesirable nucleic acid species…i.e., blocking oligonucleotide can specifically binds to a sequence in the middle portion of the undesirable nucleic acid species…the blocking oligonucleotide can specifically bind to within 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 1,000 nt from the middle point of at least one of the one or more undesirable nucleic acid species, which encompasses annealing to at least two or more different regions that are evenly or unevenly distributed regions (see para [0049]-[0056]) , thereby meeting the limitations of Applicants’ claims 11-12.
Shum et al. teach that an "undesirable nucleic acid species'' refers to a nucleic acid species that is present, e.g., in high amount, in a sample, for example the nucleic acid species representing 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20'%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more, or a range between any two of these values of the nucleic acid content in the sample (see para[0047]), and the specifically binding between the blocking oligonucleotide and the undesirable nucleic acid species can reduce the amplification and/or extension of the undesirable nucleic acid species by at least l 0%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more (see para [0050]), therefore, it is inherent that about 0.1 pmol to about 50 pmol per blocking oligonucleotide is used, thereby meeting the limitations of Applicants’ claim 19.
Shum et al. further teach that the oligonucleotide probes (i.e., primer) can comprise stochastic barcodes (see para [0088]), which can comprise a non-specific target nucleic acid sequence (i.e., random), which can be a random dimer, trimer, quatramer, pentamer, hexamer, septamer, octamer, nonamer, decamer (see para [0102]), which is also interpreted to encompass a fragmented RNA molecule, thereby meeting the limitations of Applicants’ claims 21 and 22.
Shum et al. also teach that “sample” includes, tissues, organs or organisms, which comprise whole blood, serum or plasma (see para [0033]), thereby meeting the limitations of Applicants’ claim 23.
Shum et al. further teach a method to correct for such a bias in PCR product is Molecular Indexing; however, high expressers such as ribosomal protein mRNAs, mitochondrial mRNAs, or housekeeping genes often dominate the sequencing run with little contribution to the experimental interpretation.
Krjutskov et al. teach a method for inhibiting cDNA synthesis of one or more unwanted globin mRNA (gmRNA hereafter) species in a whole-blood human, mouse, rat RNA samples during reverse transcription, comprising:
(a) providing the whole-blood RNA sample that comprises one or more desired RNA species and one or more unwanted gmRNA species,
(b) annealing 5 to 6 differently designed GlobinLock blocking oligonucleotides (GL hereafter) comprising LNA or ZNA at 3’ ends (see Figure 1a and Figure 2d) with linear GLs comprising up to 30bp (see “GlobinLock design” on page 5) to one or more regions of the one or more unwanted gmRNA species in the whole-blood RNA sample to generate a template mixture, wherein the one or more GLs are complementary, and stably bind, to the one or more regions of the one or more unwanted gmRNA species, and comprise 3' modifications such as LNA or ZNA (see Figure 1) that prevent the one or more blocking oligonucleotides from being extended, and (c) incubating the template mixture with a reaction mixture that comprises: (i) at least one reverse transcriptase, (ii) one or more reverse transcription primers, and (iii) a reaction buffer comprising Tris-HCl (salt) and KCl (see under “GlobinLock efficiency by quantitative PCR (qPCR)” on page 5), under conditions sufficient to synthesize cDNA molecules using the one or more desired RNA species as template(s), wherein cDNA synthesis using the one or more unwanted gmRNA species is inhibited (see sections “Methods”, especially under headings “GlobinLock design”, “GlobinLock efficiency by quantitative PCR (qPCR)”), thereby meeting the limitations of Applicants’ claims 2, 4-6, 8, 15-17, 23 and 27.
Krjutskov et al. teach GL used as a control didn’t have any modified nucleotides, thereby meeting the limitations of Applicants’ claim 3.
Krjutskov et al. further teach that the number of unwanted RNA species is 2, one being alpha globin mRNA and the other being beta globin mRNA (see Figure 2b), thereby meeting the limitations of Applicants’ claim 13.
Krjutskov et al. further teach the salt concentration of 0.05 μl of 1M Trix-HCL in 4 μl reaction volume is 12.5 mM, thereby meeting the limitations of Applicants’ claim 18.
Krjutskov et al. further teach that 5 μM GL was used to 30 ng/μl of human wbRNA sample (see under “GlobinLock effect by RNA-seq” on page 5 which is encompassed by about 50 pmol (italicized for added emphasis), thereby meeting the limitations of Applicants’ claim 19.
Krjutskov et al. teach synthesizing double stranded cDNA from complementary strands of the cDNA molecules, amplifying the double stranded cDNA molecules to construct a sequencing library, and sequencing the one or more desired RNA species using the sequencing library constructed (see under “GlobinLock effect by RNA-seq”, “RNA-seq data analysis” and “GlobinLock design for other species and Sanger re-sequencing” on pages 5-6), thereby meeting the limitations of Applicants’ claims 24-26.
Krjutskov et al. further teach that the RNA sample was incubated with GL for 10 min at 60 degrees Celsius and it was cooled to 42 degrees Celsius for 60 min (see last line on page 5).
QIAGEN OneStep RT-PCR handbook teaches a RT-PCR protocol wherein one performs an annealing step between 50-68 degrees Celsius for 0.5-1 min (see page 13, Table 3 and “Note”).
Becker et al. teach a method of performing RT-PCR using at least two blocking primers that anneal to different regions (see Figures 2-A to 2-D) which reduces the amplification of side products.
Chim et al. teach a method of performing RT-PCR wherein bacterial 16S rRNA is an unwanted species (see column 31, line 49; column 26, line 46)
It would have been obvious to a person of ordinary skill in the art (POSITA) prior to the effective filing date of the claimed invention to practice the RT-PCR methods taught by Vries et al., and [1] modify the annealing temperature to 50-68 degrees Celsius for 0.5-1 min then cooling to ~40 degrees Celsius prior to adding a reverse transcriptase as taught by Qiagen, in order allow sufficient time for blocking oligonucleotides to anneal and stabilize; [2] use BOs so that the entire regions of the unwanted rRNAs are evenly covered; [3] the use of salt of KCl and its concentrations during RT-PCR; and [4] obtain the RNA sample is from whole blood, serum or plasma, as taught by Shum et al., and Krjutskov et al. A POSITA would have been motivated to practice such methods in order to [a] optimize the temperature range for maximum annealing of the blocking oligonucleotides prior to reverse transcription reaction so that amplification of the unwanted RNA species is minimized; [b] optimize the reaction buffer for RT-PCR with a salt or KCl for improving catalysis, [c] design BOs in order to cover the entire rRNAs evenly so as to prevent the unwanted RNA species from any and all rRNA; and [d] obtain samples from whole blood of a subject so that one can determine if the subject has been infected with any virus. One would have had a reasonable expectation of success to practice such methods because all of the biochemical reagents and techniques for performing these types of RT-PCR were rampantly used prior to the filing of the instant application, as evidenced by Vries et al., Shum et al., Krjutskov et al., QIAGEN OneStep RT-PCR handbook, Becker et al. and Chim et al.. For the reasons provided herein, the invention as claimed is prima facie obvious over the combined teachings of the prior art.
Applicants’ Arguments:
Applicants argue that none of the cited references teaches or suggests annealing two or more blocking oligonucleotides to different regions of at least one unwanted RNA species, let alone specify the distance between two neighboring regions of the unwanted RNA species to which the two or more blocking oligonucleotides anneal. Such deficiencies of D1 and D2 have not been remedied by D3 (see 2nd paragraph on page 13). Applicants also have provided an affidavit in order to show that there was a statistically significant improvement increasing the number of readouts when using 10 BOs versus 5 BOs.
Examiner’s Explanations:
Applicants’ arguments have been fully considered but are not deemed persuasive for the following reasons. First, Applicants’ argument that the cited references do not teach annealing of two or more of the blocking oligonucleotides (BO) to different regions on the same unwanted species, is moot because teachings of Vries et al. meet this claim limitation (see above).
Furthermore, Applicants’ affidavit showing that using 5 BOs versus 10 BOs showed statistically significant increase in reducing unwanted RNA species is moot because Vries et al., Shum et al., and Krjutskov et al. all teach use of more than 10 BOs, i.e., Vries et al. specifically use 10 μM concentration of the BOs while Shum et al. teach the use of at least 1, at least 2, at least 5, at least 10, at least 100, at least 1,000 or more of the BOs that bind to one or more undesirable nucleic acid species, and therefore, it would have been obvious to a POSITA that increasing the number of BOs would improve the reduction of unwanted RNA species.
For the reasons provided herein, the invention as claimed is prima facie obvious over the combined teachings of the prior art.
Conclusion
Claims 1-8, 11-27 and 62-68 are rejected for the reasons as stated above. Applicants must respond to the objections/rejections in this Office action to be fully responsive in prosecution.
The instant Office action is non-final.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAE W LEE whose telephone number is (571)272-9949. The examiner can normally be reached on M-F between 9:00-6:00.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath 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.
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/JAE W LEE/
Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656