DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The amendment filed on 11/10/2025 has been entered. Claim 7 is cancelled. Claims 1-6 and 8-15 are pending in this application. Claims 10-15 are withdrawn. Claims 1-6, 8, and 9 are currently under examination.
Priority
This application is a 371 of PCT/EP2020/060600 filed on 04/15/2020 and claims foreign priority of EP 19169263.1 filed on 04/15/2019.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 365(c) or 386(c) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. EP 19169263.1, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Claim 5 recites “lysate is further clarified… by filtration”, which is not disclosed or supported by the prior-filed Application No. EP 19169263.1. Thus, the priority date of claim 5 is 04/15/2020.
Withdrawn Claim Objections/Rejections
The objection of claims 1 and 5 because of improper recitation, as set forth on page 4 of the Non-Final Rejection mailed on 06/13/2025, is withdrawn in view of amended claims.
The rejection of claims 1-5, 8, and 9 under 35 U.S.C. 102(a)(1) as being anticipated by Omarov et al. as evidenced by Bio-Gel® HT, as set forth on pages 4-6 of the Non-Final Rejection mailed on 06/13/2025, is withdrawn in view of amended claim 1. Claims 2-5, 8, and 9 depend from claim 1.
Claim Objections
Claim 2 remains objected to because of the following informalities: In claim 2, change the incorrect “allowing the fixation of nucleic acids” (line 3) to “for binding of the non-RISC associated nucleic acids” because fixation can mean preservation in biology and the nucleic acids encompass RISC-associated sRNAs. Appropriate correction is required.
Claim Rejections - 35 USC § 102
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.
Claims 1-3, 5, 8, and 9 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Rudel et al. (RNA 14:1244–1253, 2008, hereinafter referred to as Rudel ‘2008).
With regard to structural limitations “a method comprising the following steps: a) providing a RNA induced silencing complex (RISC)-containing sample derived from a human biological specimen (or generated by in vitro or in vivo production); b) lysing the sample using a lysis buffer to produce a lysate (or further clarified by centrifugation); c) selectively removing non RISC-associated nucleic acids from the lysate (or by loading the lysate onto a column comprising a resin for binding of the non-RISC associated nucleic acids); and d) collecting RISCs comprising RISC-associated sRNAs (or by applying an elution buffer to the column)” (claims 1-3, 5, and 8), and “e) removing the protein content from the collected RISCs using phenol/chloroform/isoamyl-alcohol extraction or proteinase K treatment” (claim 9):
Rudel ‘2008 disclosed a protocol for native elution of Ago2 from the anti-Ago2(11A9) antibody matrix. Flag/HA-tagged Ago 2 (FH-Ago2) is expressed in HEK 293 cells and immunoprecipitated with anti-Ago2(11A9). After stringent washing, a peptide encompassing the epitope of anti-Ago2(11A9) was added to compete for antibody binding. A considerable amount of bound Ago2 was eluted from the column. Similar results were obtained when endogenous Ago2 was immunoprecipitated from wild-type HEK 293 cells. To investigate if RISC activity is maintained during the above-mentioned elution procedure, peptide-eluted RISC was incubated with a target RNA complementary to endogenous miR-19b. Strikingly, only in the reaction where eluted RISC was present was a specific cleavage product detectable, demonstrating that Ago2 complexes can be efficiently eluted by peptide competition. (page 1248, left col., para. 1; right col., para. 1). Extract preparations: For total cell extract, cells were scraped off the culture plates in lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% Nonidet P-40; 2 mM EDTA; 0.5 mM DTT; 1 mM NaF; and 1 mM Pefablock [Roche]) and centrifuged for 10 min at 17,000g. Native RISC was captured with anti-Ago2(11A9) bound to protein G sepharose during a standard immunoprecipitation procedure and stringently washed: Twice with IPB containing 0.5 M NaCl, followed by a 40-min incubation in the presence of 360 mM NaCl at 4°C under rotation, and one final washing with 0.5 M NaCl. RISC precipitates were equilibrated in PBS and eluted with 100 mg of synthetic Ago2 peptide in IPB per 25 mL of beads at 25°C and 600 rpm for 90 min. Eluate was collected with a polyprep column (Biorad) equilibrated with IPB. Coimmunoprecipitated RNA was isolated by Proteinase K (AppliChem) treatment, followed by two extraction steps using first acidic phenol and second chloroform. For RNA precipitation, aqueous phase was mixed with three volumes of absolute ethanol (page 1250, right col., para. 3 and 8; page 1251, left col., para. 1 and 5).
Thus, these teachings of Rudel ‘2008 anticipate Applicant’s claims 1-3, 5, 8, and 9.
Applicant’s Arguments/Remarks filed on 11/10/2025 have been fully considered. Applicant argued “shown in Rudel et al. (Fig.1 ), the anti-Ago2(11A9) is Ago2 specific and cannot bind the tested Ago 1, 3, or 4… The IP method cannot be used to perform complete screenings of functional sRNA from RISC” (page 5, para. 3 to 4).
In response, these arguments are found not persuasive because of the following reasons. The claimed method is very broad, especially using open-ended “comprising”, and does not exclude Ago2 specific antibody or immunoprecipitation (IP). Thus, the teachings of Rudel ‘2008 anticipate claimed method, as described above. To overcome the anticipation rejection, Applicant may include additional limitation that is not taught by the reference. Any amendment after Final may not be entered if new issue arises and/or extensive search is required.
New (necessitated by amendment)/Claim Rejections - 35 USC § 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 factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
(I) Claims 1-3, 5, 6, 8, and 9 remain rejected under 35 U.S.C. 103 as being unpatentable over Rudel et al. (RNA 14:1244–1253, 2008, hereinafter referred to as Rudel ‘2008). Claims 1-3, 5, 8, and 9 are rejected here because they have been rejected under 102 above. Thus, the above disclosure of Rudel ‘2008 is incorporated in its entirety here.
Rudel ‘2008 did not explicitly disclose the limitation “the column is a 96 well plate or a microfluidic chip”, required by claim 6.
However, Rudel ‘2008 also disclosed an ELISA screen coated with the injected peptide. HeLa cells were reverse transfected with siRNAs in six-well plates (page 1246, left col. para. 1; page 1251, right col., para. 2), suggesting that a multi-well or a high-throughput device is used for screening.
Thus, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to substitute the six-well plate as taught by Rudel ‘2008 with either a 96-well plate or a microfluidic chip in common laboratory setting to increase throughput of the immunoprecipitation method. Thus, one of skill in the art would have a reasonable expectation that by substituting the six-well plate as taught by Rudel ‘2008 with either a 96-well plate or a microfluidic chip in common laboratory setting to increase throughput of the immunoprecipitation method, one would achieve Applicant’s claims 1-3, 5, 6, 8, and 9. "Exemplary rationales that may support a conclusion of obviousness include: (B) Simple substitution of one known element for another to obtain predictable results". See MPEP § 2143 [R-01.2024] [I].
Applicant’s Arguments/Remarks filed on 11/10/2025 have been fully considered. Applicant argued “the immunoprecipitation methods disclosed in Rudel cannot be used to perform complete screenings of functional sRNA from RISC” (page 5, para. 6).
In response, these arguments are found not persuasive because of the following reasons. The claimed method is very broad, especially using open-ended “comprising”, and does not exclude immunoprecipitation (IP). See also the response under 102 above. To overcome the obviousness rejection, Applicant may include additional limitation that is not taught or suggested by the reference. Any amendment after Final may not be entered if new issue arises and/or extensive search is required
(II) Claims 1-6, 8, and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Rudel et al. (RNA 14:1244–1253, 2008, hereinafter referred to as Rudel ‘2008), as applied to claims 1-3, 5, 6, 8, and 9, in view of Omarov et al. (Virology 490:41–48, 2016, hereinafter referred to as Omarov ‘2016) as evidenced by Bio-Gel® HT (Bio-Rad, Introduction to Multimodal or Mixed-Mode Chromatography, accessed in June 2025) .
Rudel ‘2008 did not explicitly disclose the limitation “the resin is an anion exchange resin for binding of the non-RISC associated nucleic acids”, required by claim 6
Omarov ‘2016 disclosed that N. benthamiana leaf tissue infected with TdP19 was homogenized in 50ml loading buffer(10mM sodium phosphate, pH6.8). Subsequently, the extract was filtered through cheesecloth followed by centrifugation for 15min at 14,000g at 4 °C. The supernatant (100ml) was loaded onto a 12 x 3.5 cm2 column packed with hydroxyapatite Bio-Gel HT (Bio-Rad,Hercules, CA). The column was washed with 300ml loading buffer and the bound proteins were subsequently eluted with a gradient of increasing concentrations of sodium phosphate buffer (10 – 200mM, pH6.8). Antiviral RISC (vRISC)-active fractions were obtained upon hydroxyapatite chromatography fractions. To conduct siRNAs dissociation experiments, antiviral RISC (vRISC) active fractions obtained after hydroxyapatite chromatography were fractionated on a same column that was pre-equilibrated with elution buffer (50mM Tris–HCl, pH7.4) with the addition of 200mM NaCl. To dissociates iRNAs from protein complexes, 300 ml aliquots of each fraction obtained by the chromatography procedures were treated with 10% SDS (30 ml) at 65 °C for 15 min, followed by phenol/chloroform extraction. RNA was subsequently precipitated with 2.5 volumes of 100% ethanol followed by centrifugation and resuspension in10 ml of loading buffer (page 47, left col., para. 3 to 5). Bio-Gel® HT (cited here as evidence only) disclosed that Hydroxyapatite, Ca10(PO4)6(OH)2, is a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C-sites) and pairs of –OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Hydroxyapatite (CHT/CFT/Bio-Gel HT/HTP) is a mixed-mode medium. When using a mixed-mode ligand containing both hydrophobic and ionic elements, increasing ionic strength will disrupt ionic bonds, however, increasing salt concentration will promote hydrophobic interactions (page 3/8, para. 1; page 2/8, para. 2 and 5).
Thus, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to substitute the protein G sepharose for binding with anti-Ago2(11A9) as taught by Rudel ‘2008 with hydroxyapatite Bio-Gel HT to serve as anion exchange in view of Omarov ‘2016 as evidenced by Bio-Gel® HT to capture any RISC complex, as described above. Thus, one of skill in the art would have a reasonable expectation that by substituting the protein G sepharose for binding with anti-Ago2(11A9) as taught by Rudel ‘2008 with hydroxyapatite Bio-Gel HT in view of Omarov ‘2016 as evidenced by Bio-Gel® HT, one would achieve Applicant’s claims 1-6, 8, and 9. "Exemplary rationales that may support a conclusion of obviousness include: (B) Simple substitution of one known element for another to obtain predictable results". See MPEP § 2143 [R-01.2024] [I].
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/YIH-HORNG SHIAO/Primary Examiner, Art Unit 1691