DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
2. Applicant's election with traverse of tRNA (and further tRNA-derived fragments/tRNA-derived RNAs) in response to the first species election, species (a) – treating with (i)(a) a polynucleotide kinase and an RNA ligase in response to the second species election and extracellular sample (and further blood serum) in response to the third species election in the reply filed on 11 May 2026 is acknowledged. The traversal is on the grounds that the species are so related that the Examiner could search and examine them together with undue burden. This is not found persuasive because the range of RNA types claimed and the difference in enzymatic function and substrate requirements of the enzymes claimed are so different as to require different fields of search.
However, upon further consideration the election requirement of the THIRD species is determined to be searchable without additional undue burden, and therefore the election requirement for the THIRD species is withdrawn.
The requirement is still deemed proper for the elections of the FIRST and SECOND species and is therefore made FINAL.
Claim Rejections - 35 USC § 102
3. The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
4. Claims 1-3, 6-8, 11, 14, 15, 19, 22, 23, 31, 32, 34-36, 40-42, 45, 48, 49, 55, 56 and 57 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Honda et al (Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method, Nature Protocols, 11, 3, published 11 February 2016). The rejections of claims 1, 15, 31 and 49 are evidenced by Meridian (Isolate II RNA Mini Kit, Product Manual).
Regarding claim 1, Honda teaches a method of enzymatically repairing RNAs that are nicked or at least partially cleaved (abstract and FIG 2). Honda teaches providing BT-474 breast cancer cells that comprise 5'-tRNA halves (i.e., a biological sample containing RNAs that are nicked or at least partially cleaved; FIG 2 and pg. 477 column 2 ¶ 1). Honda teaches the total extraction of RNAs (i.e., removing non-RNA components) using Bioline cat. No. BIO-38032. This kit does not use denaturing reagents such as heat or phenol, as evidenced by Meridian (pg. 7 – Kit Components and pg. 9 ¶ 2). It is noted that Honda does require a furth denaturing purification of the total RNA extract (pg. 478 column 1 ¶ 4), however this claim as written does NOT exclude the possibility of a denaturing step (such as the limitations of claims 16-18), merely that a non-denaturing purification is performed. Therefore, Honda is considered to read on this limitation. Honda additionally teaches the removal of the 3' cyclic phosphate by treatment with T4 PNK (i.e., a polynucleotide kinase; pg. 478 column 2 ¶ 1) and the ligation of an RNA adapter to the converted 3' end (i.e., treatment with an RNA ligase; pg. 478 column 2 ¶ 2 and pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase). Based on the language of the claim (thereby forming repaired RNAs from the nicked or at least partially cleaved RNAs), any RNA purified and treated in the fashion of claim 1 is considered “forming a repaired RNA.”
Regarding claims 2 and 3, Honda teaches the nicked or at least partially cleaved RNAs are tRNA halves (i.e., tRNA fragments pg. 477 column 2 ¶ 2).
Regarding claim 6, Honda teaches that the treating is with PNK and an RNA ligase (pg. 478 column 2 ¶ 1 and pg. 478 column 2 ¶ 2 and pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase).
Regarding claim 7, Honda teaches that the polynucleotide kinase is followed by the addition of the RNA ligase (pg. 479 column 2 ¶ 1 and ¶ 2).
Regarding claim 8, Honda teaches that the polynucleotide kinase is T4 PNK (pg. 479 column 2 ¶ 1).
Regarding claim 11, Honda teaches that the RNA ligase is RNA ligase 1 (pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase).
Regarding claim 14, Honda teaches that the treating is carried out at least in part in the presence of ATP (pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase).
Regarding claim 15, Honda teaches that the total RNA extraction comprises a solid phase extraction (as evidenced by Meridian pg. 8).
Regarding claims 19, 22 and 23, Honda teaches that the biological sample is a cancer cell (pg. 477 column 2 ¶ 2).
Regarding claim 31, Honda teaches a method of enzymatically repairing RNAs that are nicked or at least partially cleaved (abstract and FIG 2). Honda teaches providing BT-474 breast cancer cells that comprise 5'-tRNA halves (i.e., a biological sample containing RNAs that are nicked or at least partially cleaved; FIG 2 and pg. 477 column 2 ¶ 1). Honda teaches the total extraction of RNAs (i.e., removing non-RNA components) using Bioline cat. No. BIO-38032. This kit does not use denaturing reagents such as heat or phenol, as evidenced by Meridian (pg. 7 – Kit Components and pg. 9 ¶ 2). It is noted that Honda does require a furth denaturing purification of the total RNA extract (pg. 478 column 1 ¶ 4), however this claim as written does NOT exclude the possibility of a denaturing step (such as the limitations of claims 16-18), merely that a non-denaturing purification is performed. Therefore, Honda is considered to read on this limitation. Honda additionally teaches the removal of the 3' cyclic phosphate by treatment with T4 PNK (i.e., a polynucleotide kinase; pg. 478 column 2 ¶ 1) and the ligation of an RNA adapter to the converted 3' end (i.e., treatment with an RNA ligase; pg. 478 column 2 ¶ 2 and pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase). Honda teaches detecting the repaired RNAs (pg. 478 column 2 ¶ 2).
Regarding claims 32 and 34, Honda teaches that the detecting comprises next-generation sequencing (i.e., high-throughput sequencing; pg. 478 column 2 ¶ 2).
Regarding claim 35, Honda teaches that the samples are treated with RNase inhibitors after the step of purifying the nicked or at least partially cleaved RNAs (pg. 483 step 46).
Regarding claims 36 and 37, Honda teaches the nicked or at least partially cleaved RNAs are tRNA halves (i.e., tRNA fragments pg. 477 column 2 ¶ 2).
Regarding claim 40, Honda teaches that the treating is with PNK and an RNA ligase (pg. 478 column 2 ¶ 1 and pg. 478 column 2 ¶ 2 and pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase).
Regarding claim 41, Honda teaches that the polynucleotide kinase is followed by the addition of the RNA ligase (pg. 479 column 2 ¶ 1 and ¶ 2).
Regarding claim 42, Honda teaches that the polynucleotide kinase is T4 PNK (pg. 479 column 2 ¶ 1).
Regarding claim 45, Honda teaches that the RNA ligase is RNA ligase 1 (pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase).
Regarding claim 48, Honda teaches that the treating is carried out at least in part in the presence of ATP (pg. 479 Materials – TruSeq small RNA library preparation kits – T4 RNA ligase).
Regarding claim 49, Honda teaches that the total RNA extraction comprises a solid phase extraction (as evidenced by Meridian pg. 8).
Regarding claims 53, 56 and 57, Honda teaches that the biological sample is a cancer cell (pg. 477 column 2 ¶ 2).
Claim Rejections - 35 USC § 103
5. 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.
6. This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
7. Claims 20, 21, 25, 26, 54, 55, 59 and 60 are rejected under 35 U.S.C. 103 as being unpatentable over Honda et al (Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method, Nature Protocols, 11, 3, published 11 February 2016), as evidenced by Meridian (Isolate II RNA Mini Kit, Product Manual) in view of Tosar et al (Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome, Nucleic Acids Research, 48, 22 published 12 August 2020).
Regarding claim 20, Honda teaches the method of claim 1 as discussed fully above and incorporated here. Honda teaches that the biological sample is a cell.
Honda does not teach that the biological sample is from an extracellular biofluid.
However, Tosar teaches that extracellular biofluids comprise tRNA halves (i.e., the same type of RNA Honda was analyzing; pg. 12874 column 2 ¶ 1 and ¶ 3).
It would have been obvious to one having ordinary skill in the art to have replaced the biological sample taught by Honda with the extracellular biofluid taught by Tosar to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Tosar specifically teaches that exRNAs are immunologically nonsilent (pg. 12883 column 1 ¶ 4) and ‘offer a window’ to new molecules with biomarker potential (12886 column 1 ¶ 3). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited reference could have been combined with predictable results because the known techniques in the cited references predictably result in the study and detection of nicked or partially cleaved RNA fragments.
Regarding claim 21, Tosar teaches that the extracellular fluid is urine, plasma or serum (pg. 12874 column 2 ¶ 1).
Regarding claims 25 and 26, Tosar teaches that the nicked or partially cleaved RNAs are formed from nonvesicular extracellular RNAs (pg. 12874 column 1 ¶ 3 and pg. 12883 column 2 ¶ 1).
Regarding claim 54, Honda teaches the method of claim 31 as discussed fully above and incorporated here. Honda teaches that the biological sample is a cell.
Honda does not teach that the biological sample is from an extracellular biofluid.
However, Tosar teaches that extracellular biofluids comprise tRNA halves (i.e., the same type of RNA Honda was analyzing; pg. 12874 column 2 ¶ 1 and ¶ 3).
It would have been obvious to one having ordinary skill in the art to have replaced the biological sample taught by Honda with the extracellular biofluid taught by Tosar to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Tosar specifically teaches that exRNAs are immunologically nonsilent (pg. 12883 column 1 ¶ 4) and ‘offer a window’ to new molecules with biomarker potential (12886 column 1 ¶ 3). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited reference could have been combined with predictable results because the known techniques in the cited references predictably result in the study and detection of nicked or partially cleaved RNA fragments.
Regarding claim 55, Tosar teaches that the extracellular fluid is urine, plasma or serum (pg. 12874 column 2 ¶ 1).
Regarding claims 59 and 60, Tosar teaches that the nicked or partially cleaved RNAs are formed from nonvesicular extracellular RNAs (pg. 12874 column 1 ¶ 3 and pg. 12883 column 2 ¶ 1).
8. Claim 33 is rejected under 35 U.S.C. 103 as being unpatentable over Honda et al (Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method, Nature Protocols, 11, 3, published 11 February 2016), as evidenced by Meridian (Isolate II RNA Mini Kit, Product Manual) in view of Barnell et al (United States Patent Application No. US 20210214797, published 15 July 2021).
Regarding claim 33, the method of claim 31 is discussed fully above and incorporated here.
Honda does not teach that the detecting comprises quantitative RT-PCR (RT-qPCR).
However, Barnell teaches the detection of RNA biomarkers (e.g., tRNA) and teaches RT-qPCR as a known alternative to RNA sequencing ([0071]).
It would have been obvious to one having ordinary skill in the art to have simply substituted the RNA sequencing method taught by Honda with the RT-qPCR method taught by Barnell to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution because Barnell specifically teaches RT-qPCR and RNA sequencing as alternatives for the detection of tRNA biomarkers. In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in RNA detection and analysis.
9. Claims 1-3, 5-8, 11, 14-19, 22, 27, 28, 31, 32, 36, 37, 39-42, 45, 49-53, 56, 61 and 62 are rejected under 35 U.S.C. 103 as being unpatentable over Amitsur et al (Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA, The EMBO Journal, 6, 8, 2499-2503, published 01 August 1987) in view of Yoshino et al (Low-cost RNA extraction method for highly scalable transcriptome studies, Breeding Science, 70, 481-486, published 03 July 2020). The rejections of claims 28 and 62 are further evidenced by Davidov et al (RloC: a wobble nucleotide-excising and zinc-responsive bacteria tRNase, Molecular microbiology, 69, 6, 1560-1574, published 07 August 2008).
Regarding claim 1, Amitsur teaches a method of enzymatically repairing RNAs that are nicked or at least partially cleaved (abstract). Amitsur teaches providing a biological sample containing RNAs that are nicked or at least partially cleaved (T4-infected E. coli wild type strains; abstract and pg. 2499 column 1 ¶ 2). Amitsur teaches treating the RNAs with a polynucleotide kinase and an RNA ligase (pg. 2499 column 2 ¶ 3). Amitsur teaches that this “repairs the anticodon nuclease cleavage” (pg. 2499 column 2 ¶ 1).
Amitsur does not teach purifying the nicked or at least partially cleaved RNAs contained in the biological sample under a non-denaturing condition to remove non-RNA components.
However, Yoshino teaches a method of RNA extraction under non-denaturing conditions to remove non-RNA components (pg. 482 column 1 ¶ 3).
It would have been obvious to one having ordinary skill in the art to have replaced the denaturing purification taught by Amitsur (pg. 2502 column 2 ¶ 5) with the non-denaturing purification taught by Yoshino to arrive at the instantly claimed invention with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to make this modification because non-denaturing purification would likely improve the recovery of nicked tRNAs without needing to rely on the fragments assembly correctly in the in vitro experiments taught by Amitsur (pg. 2503 column 1 ¶ 2) and Yoshino specifically teaches that their purification method is suitable for small RNA extraction (pg. 485 column 2 ¶ 2). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the purification and analysis of small RNAs.
Regarding claim 2, Amitsur teaches that the nicked or at least partially cleaved RNAs comprise tRNA (abstract, FIG 1).
Regarding claim 3, Amitsur teaches that the nicked or at least partially cleaved RNAs comprise tRNA fragments (FIG 1, pg. 2499 column 2 ¶ 5, pg. pg. 2503 column 1 ¶ 2).
Regarding claims 5-8, Amitsur teaches the polynucleotide kinase – RNA ligase mediate repair of damaged tRNAs (pg. 2499 column 2 ¶ 1, and that host tRNAs are cleaved and repaired by PNK and RNA ligase – abstract). In cells the PNK is considered ‘added together with’ the RNA ligase, and in vitro the PNK is added followed by the RNA ligase (pg. 2503 column 1 ¶ 2).
Regarding claim 11, Amitsur teaches an RNA ligase corresponding the bacteriophage rli gene (i.e., RNA ligase 1; abstract).
Regarding claim 14, Amitsur teaches that the treating is carried out in the presence of ATP (pg. 2503 column 1 ¶ 2).
Regarding claims 15-18, Yoshino teaches that the non-denaturing condition comprises a solid phase extraction on beads (pg. 482 column 1 ¶ 3), and that the method is carried out in the absence of a condition that results in the denaturation of an RNA molecule (i.e., there is no heat, adapter ligation, chemical denaturant, or phenol; pg. 483 column 1 ¶ 3).
Regarding claims 19 and 22, Amitsur teaches that the source of the biological sample is a bacterial cell (pg. 2502 column 2 ¶ 5).
Regarding claim 27, Amitsur teaches the “restoration of lysine tRNA” (pg. 2502 column 1 ¶ 5) and the restoration of a band corresponding to full-length lysine tRNA (FIG 2). Amitsur teaches the tRNA fragments emanate from lysine tRNA (band L1 in FIG 2) and are “subsequently repaired” to restore L1 (pg. 2500 column 1 ¶ 3).
Regarding claim 28, Amitsur teaches that the nuclease activity relies on the host E. coli gene prr (pg. 2499 column 2 ¶ 1). The tRNase associated with this gene has been shown to yield tRNA fragments 1 nucleotide shorter than expected as evidenced by Davidov (pg. 1565 column 1 ¶ 1). Therefore, some of the repaired RNAs would be 1 nucleotide shorter compared to its parent RNA.
Regarding claim 31, Amitsur teaches a method of enzymatically repairing RNAs that are nicked or at least partially cleaved (abstract). Amitsur teaches providing a biological sample containing RNAs that are nicked or at least partially cleaved (T4-infected E. coli wild type strains; abstract and pg. 2499 column 1 ¶ 2). Amitsur teaches treating the RNAs with a polynucleotide kinase and an RNA ligase (pg. 2499 column 2 ¶ 3). Amitsur teaches that this “repairs the anticodon nuclease cleavage” (pg. 2499 column 2 ¶ 1).
Amitsur does not teach purifying the nicked or at least partially cleaved RNAs contained in the biological sample under a non-denaturing condition to remove non-RNA components.
However, Yoshino teaches a method of RNA extraction under non-denaturing conditions to remove non-RNA components (pg. 482 column 1 ¶ 3).
It would have been obvious to one having ordinary skill in the art to have replaced the denaturing purification taught by Amitsur (pg. 2502 column 2 ¶ 5) with the non-denaturing purification taught by Yoshino to arrive at the instantly claimed invention with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to make this modification because non-denaturing purification would likely improve the recovery of nicked tRNAs without needing to rely on the fragments assembly correctly in the in vitro experiments taught by Amitsur (pg. 2503 column 1 ¶ 2) and Yoshino specifically teaches that their purification method is suitable for small RNA extraction (pg. 485 column 2 ¶ 2). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the purification and analysis of small RNAs.
Regarding claim 32, Amitsur teaches the detecting comprises sequencing (2502 column 2 ¶ 6) and nucleic acid hybridization (FIG 2).
Regarding claim 36, Amitsur teaches that the nicked or at least partially cleaved RNAs comprise tRNA (abstract, FIG 1).
Regarding claim 37, Amitsur teaches that the nicked or at least partially cleaved RNAs comprise tRNA fragments (FIG 1, pg. 2499 column 2 ¶ 5, pg. pg. 2503 column 1 ¶ 2).
Regarding claims 39-42, Amitsur teaches the polynucleotide kinase – RNA ligase mediate repair of damaged tRNAs (pg. 2499 column 2 ¶ 1, and that host tRNAs are cleaved and repaired by PNK and RNA ligase – abstract). In cells the PNK is considered ‘added together with’ the RNA ligase, and in vitro the PNK is added followed by the RNA ligase (pg. 2503 column 1 ¶ 2).
Regarding claim 45, Amitsur teaches an RNA ligase corresponding to the bacteriophage rli gene (i.e., RNA ligase 1; abstract).
Regarding claim 48, Amitsur teaches that the treating is carried out in the presence of ATP (pg. 2503 column 1 ¶ 2).
Regarding claims 49-52, Yoshino teaches that the non-denaturing condition comprises a solid phase extraction on beads (pg. 482 column 1 ¶ 3), and that the method is carried out in the absence of a condition that results in the denaturation of an RNA molecule (i.e., there is no heat, adapter ligation, chemical denaturant, or phenol; pg. 483 column 1 ¶ 3).
Regarding claims 53 and 56, Amitsur teaches that the source of the biological sample is a bacterial cell (pg. 2502 column 2 ¶ 5).
Regarding claim 61, Amitsur teaches the “restoration of lysine tRNA” (pg. 2502 column 1 ¶ 5) and the restoration of a band corresponding to full-length lysine tRNA (FIG 2). Amitsur teaches the tRNA fragments emanate from lysine tRNA (band L1 in FIG 2) and are “subsequently repaired” to restore L1 (pg. 2500 column 1 ¶ 3).
Regarding claim 62, Amitsur teaches that the nuclease activity relies on the host E. coli gene prr (pg. 2499 column 2 ¶ 1). The tRNase associated with this gene has been shown to yield tRNA fragments 1 nucleotide shorter than expected as evidenced by Davidov (pg. 1565 column 1 ¶ 1). Therefore, some of the repaired RNAs would be 1 nucleotide shorter compared to its parent RNA.
Conclusion
10. Claims 29, 30, 63 and 64 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
11. Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN ELLIS YOUNG whose telephone number is (703)756-5397. The examiner can normally be reached M-T 0800 - 1630.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
Prosecution Insights