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
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Status of Claims
Claims 1, 3-20, 25, 51 and 52 are currently pending in the instant application. Claims 1, 3-16, 18-20 and 25 have been amended by Applicants’ amendment filed 07-13-2025. Claims 51 and 52 have been added by Applicants’ amendment filed 07-13-2026. No claims have been canceled by Applicants’ amendment filed 07-13-2026.
A complete reply to the final rejection must include cancellation of nonelected claims or other appropriate action (37 CFR 1.144) See MPEP § 821.01.
Therefore, claims 1, 3-20, 25, 51 and 52 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed April 25, 2023, is a 35 U.S.C. 371 national stage filing of International Application PCT/US2021/056471, filed October 25, 2021, which claims the benefit of US Provisional Patent Application 63105741, filed October 26, 2020.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on July 13, 2026 has been considered. An initialed copy of the IDS accompanies this Office Action.
Withdrawn Objections/Rejections
Applicants’ amendment and arguments filed July 13, 2026 are acknowledged and have been fully considered. The Examiner has re-weighed all the evidence of record. Any rejection and/or objection not specifically addressed below are herein withdrawn.
Claim Objections
The rejection of claims 1, 4-6, 11, 13, 15, 16 and is withdrawn because Applicant has amended claims 1, 4-6, 11, 13, 15, 16 and 25 to spell out or delete abbreviations including “PCR”, "UV”, “UDP”, “mRNA”, “bp”, “UTR”, “LNA”, “APP”, “ATG9A”, “BTG2” and ULK1”.
In view of the withdrawn objection, Applicant’s arguments are rendered moot.
The objection to claim 5 because claim 5 recites the terms: “prionaldehyde” and “water-soluble carbomiidides” because Applicant has amended the claim to provide the correct spelling and/or to delete a term.
In view of the withdrawn objection, Applicant’s arguments are rendered moot.
Specification Objection
The objection to the disclosure is withdrawn because Applicant has amended the Specification in the reply filed July 13, 2026.
In view of the withdrawn objection, Applicant’s arguments are rendered moot.
Claim Rejections - 35 USC § 102
The rejection of claims 1, 3, 4, 6-10, 12, 13, 15-17 and 19 is withdrawn under 35 U.S.C. 102(a1)/102(a2) as being anticipated by Hong, Hung (hereinafter “Hong”) (Thesis, The Hong Kong Polytechnic University, 2017, 1-399) as evidenced by Van Der Oost et al. (hereinafter “Van Der Oost”) (US Patent No. 9902973, issued February 27, 2018); and Fotin-Mleczek et al. (hereinafter “Fotin-Mleczek”) (US Patent No. 11078247, issued Augst 3, 2021; PCT/EP2017/060692, filed May 4, 2017).
Hong does not specifically exemplify the limitations as arranged.
In view of the withdrawn rejection, Applicant’s arguments are rendered moot.
Maintained Objections/Rejections
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1, 3-20 and 25 is maintained, and claims 51 and 52 are newly rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Claims 1, 7 and 8 are indefinite for the recitation of the term “the complexes” such as recited in claim 1, line 5. There is insufficient antecedent basis for the term “the complexes” in the claim because claim 1, line 4 recites the term “Ago2-miRNA-target RNA complexes.”
Claims 1, 12 and 51 are indefinite for the recitation of the term “the miRNA molecules” such as recited in claim 1, line 8. There is insufficient antecedent basis for the term “the miRNA molecules” in the claim.
Claim 1 is indefinite for the recitation of the term “performing an enriching step comprising contacting the PCR products with miRNA-specific hybridization probes to selectively enrich for PCR products comprising the miRNA of interest” as recited in claim 1, lines 17-18 because the as-filed Specification and/or the original claims do not recite the limitation of enriching after PCR and, thus, the metes and bounds of the claim cannot be determined.
Claim 1 is indefinite for the recitation of the terms “thereby forming enriched PCR products comprising the miRNA of interest,” and “thereby forming enriched PCR products…and non-chimeric RNA molecules” such as recited in claim 1, lines 19-22 because it is unclear how the step of “performing an enriching step” as recited produces two different results such that it is unclear whether the step produces whether the step produces one or the other of the PCR products, or whether the step is meant to product both products. Moreover, it is unclear how PCR products comprising chimeric RNA molecule and non-chimeric RNA molecules (lines 15-16) can form three different enriched products that include the miRNA of interest (e.g., which is not recited to be a PCR product) and, thus, the metes and bounds of the claim cannot be determined.
Claim 1 is indefinite for the recitation of the term “the sequencing data” such as recited in claim 1, line 26. There is insufficient antecedent basis for the term “the sequencing data” in the claim.
Claim 1 is indefinite for the recitation of the term “the subject” such as recited in claim 1, line 27. There is insufficient antecedent basis for the term “the subject” in the claim.
Claim 3 is indefinite for the recitation of the term “wherein the RNA sample is obtained from cells or tissue, the method further comprising lysing the cells or tissue prior to isolating” such as recited in claim 3, lines 1-3 because claim 3 depends from instant claim 1, wherein amended claim 1 already recites that the RNA sample is obtained from cells and tissues, and that these are lysed. Additionally, lysing must occur prior to isolating the complexes because that is the only way that isolating the Ago2-miRNA-target RNA complexes can be carried out. Claim 3 merely repeats the same process as recited in instant claim 1 and, thus, the metes and bounds of the claim cannot be determined.
Claim 9 is indefinite for the recitation of the term “to identify and quantify miRNA-target interactions” such as recited in claim 9, lines 2-3 because claim 9 depends from claims 1, 7 and 8, wherein claims 1, 7 and 8 do not recite any miRNA-target RNA interactions. Moreover, it is unclear how sequencing the PCR products will quantify an interaction (e.g., a behavior, communication, influence, etc.) and, thus, the metes and bounds of the claim cannot be determined.
Claims 11, 12, 14, 16, 51 and 52 are indefinite for the recitation of the term “the probes” such as recited in claim 11, line 1. There is insufficient antecedent basis for the term “the probes” in the claim because claim 1 recites the term “miRNA-specific hybridization probes.” The Examiner suggests that Applicant amend the claim to recite, for example, “wherein the miRNA-specific hybridization probes are anti-sense nucleic acid probes.”
Claim 13 is indefinite for the recitation of the term “the target RNA portion” such as recited in claim 13, line 1. There is insufficient antecedent basis for the term “the target RNA portion” in the claim. Moreover, claim 13 depends from instant claim 1, wherein claim 1 does not recite a target RNA portion, mapping to genes, and/or mapping to untranslated region of genes and, thus, the metes and bounds of the claim cannot be determined.
Claim 18 is indefinite for the recitation of the terms “chimeric reads” and “uniquely mapped reads” such as recited in claim 18, line 2 because claim 18 depends from instant claim 1, wherein claim 1 does not recite the presence of chimeric reads, a step of mapping reads, and/or uniquely mapped reads and, thus, the metes and bounds of the claim cannot be determined.
Claims 19 and 20 are indefinite for the recitation of the terms “increase” and “increased such as recited in claim 18, line 2 because the terms “increase” and “increased” are relative terms that render the claim indefinite. The terms “increase” and “increased” are not defined by the claim, and the Specification does not provide a standard for ascertaining the requisite proportion of chimeric reads and/or overall chimeric read population as compared to some other value that qualifies as an “increase” in the population of chimeric reads, such that one of ordinary skill in the art would not be reasonably appraised of the scope of the invention.
Claim 19 is indefinite for the recitation of the term “the proportion” such as recited in claim 19, line 2. There is insufficient antecedent basis for the term “the proportion” in the claim.
Claim 19 is indefinite for the recitation of the term “the proportion of chimeric reads” such as recited in claim 19, line 2 because claim 19 depends from instant claim 1, wherein claim 1 does not recite the presence of chimeric reads, or a proportion of chimeric reads in the enriched PCR products and, thus, the metes and bounds of the claim cannot be determined.
Claim 20 is indefinite for the recitation of the term “the overall chimeric read population” such as recited in claim 20, lines 1-2. There is insufficient antecedent basis for the term “the overall chimeric read population” in the claim. Moreover, claim 20 depends from instant claims 1 and 19, wherein claims 1 and 19 do not recite the presence of an overall chimeric read population because claim 19 recites the term “increases the proportion of chimeric reads.”
Claim 51 is indefinite for the recitation of the terms “reads”, “two or more distinct miRNA molecules”, “two or more targeted miRNA molecules”, and “total chimeric library” such as recited in claim 51, lines 2-4 because claim 51 depends from instant claim 1, wherein claim 1 does not recite the presence of reads, two or more distinct miRNA molecules, two or more targeted miRNA molecules, and/or a total chimeric library and, thus, the metes and bounds of the claim cannot be determined.
Claim 52 is indefinite for the recitation of the terms “the enriched RNA molecules” and “the beads” such as recited in claim 52, line 3. There is insufficient antecedent basis for the terms “the enriched RNA molecules” and “the beads” in the claim because claim 52, line 2 recites the term “streptavidin beads”. The Examiner suggests that Applicant amend the claim to recite, for example, “washing the streptavidin beads.”
Claims 2, 4-6, 15, 17 and 25 are indefinite insofar as they ultimately depend from instant claim 1.
Claim Rejections - 35 USC § 112(d)
The rejection of claims 3, 9, 13 and 18-20 is maintained, and claim 51 is newly rejected under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 3 recites (in part): “wherein the RNA sample is obtained from cells or tissue, the method further comprising lysing the cells or tissue prior to isolating the Ago2-miRNA-target RNA complexes” in lines 1-3 because claim 3 depends from instant claim 1, wherein claim 1 already recites that the RNA sample is obtained from cells and tissues, and that these are lysed. Additionally, isolating the Ago2-miRNA-target RNA complexes fundamentally requires initially lysing a cell. Thus, claim 3 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 9 recites (in part): “the PCR products are subjected to the sequencing to identify and quantify miRNA-target RNA interactions” in lines 1-3 because claim 9 depends from instant claim 1, wherein claim 1 does not recite the any type or quantity of miRNA-target RNA interactions. Thus, claim 9 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 13 recites (in part): “wherein the target RNA portion of the chimeric RNA molecules maps to specific genes or 3’-UTR of genes” in lines 1-3 because claim 13 depends from instant claim 1, wherein claim 1 does not recite a target RNA portion, mapping to genes, and/or mapping to untranslated region of genes. Thus, claim 13 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 18 recites (in part): “wherein the enriching step produces about 5% to about 35% chimeric reads in the enriched PCR products” in lines 1-2 because claim 18 depends from instant claim 1, wherein claim 1 does not recite the presence of chimeric reads, a step of mapping reads, and/or uniquely mapped reads. Thus, claim 18 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 19 recites (in part): “the proportion of chimeric reads” in line 2 because claim 19 depends from instant claim 1, wherein claim 1 does not recite the presence of chimeric reads, or a proportion of chimeric reads in the enriched PCR products. Thus, claim 19 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 20 recites (in part): “wherein the overall chimeric read population is increased” in lines 1-2 because claim 20 depends from instant claims 1 and 19, wherein claims 1 and 19 do not recite the presence of an overall chimeric read population. Thus, claim 20 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 51 recites (in part): “wherein the enriching step uses a plurality of antisense probes…a total chimeric library without the enriching step” in lines 1-4 because claim 51 depends from instant claim 1, wherein claim 1 does not recite the presence of reads, two or more distinct miRNA molecules, two or more targeted miRNA molecules, and/or a total chimeric library. Thus, claim 51 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Claim Rejections - 35 USC § 103
The rejection of claims 1, 3-20 and 25 is maintained, and claims 51 and 52 are newly rejected, under 35 U.S.C. 103 as being unpatentable over Hong, Hung (hereinafter “Hong”) (Thesis, The Hong Kong Polytechnic University, 2017, 1-399; of record) in view of Xu et. al. (hereinafter “Xu”) (RNA Biology, 2017, 14(2), 259-274; of record) as evidenced by Van Der Oost et al. (hereinafter “Van Der Oost”) (US Patent No. 9902973, issued February 27, 2018; of record); and Fotin-Mleczek et al. (hereinafter “Fotin-Mleczek”) (US Patent No. 11078247, issued Augst 3, 2021; PCT/EP2017/060692, filed May 4, 2017; of record).
Regarding claims 1, 4, 6-8, 10, 15 and 17, Hong teaches that the RNA Interactome Identification by Next Generation Sequencing (RIINGS) experiment starts with cells or tissues that have been disrupted into small pieces, wherein like other RNA intercome identification methods RIINGS requires crosslinking, immuno-precipitation and RNA digestion (interpreting cells and tissues as an RNA sample comprising mRNA or mRNA fragments; crosslinking; disruption produces RNA fragments; and IP as isolating, claims 1, 4, 6 and 7) (pg. 99, Section 1.6.1, first full paragraph). Hong teaches the Dicer-cleaved product is eventually loaded onto an Argonaute protein to form the core of the effector complex called RNA-induced silencing complex (RISC) (interpreted as an Ago2-miRNA-target RNA complex comprising a Ago2 protein, claim 1) (pg. 30, first partial paragraph, lines 13-14). Hong teaches that crosslinking can be achieved by 254 nm UV light or 365 nm UV light with the assistance of 4-thiouridine (4SU) (interpreted as crosslinking with UV light can include a chemical agent 4SU, claim 4) (pg. 99, Section 1.6.1; last partial paragraph, lines 1-2), wherein it is known in the art that cells or complexes can be fixed or crosslinked using suitable fixatives and crosslinkers including: formaldehyde, glutaraldehyde, ethanol-based fixatives, methanol-based fixatives, acetone, acetic acid, osmium tetraoxide, potassium dichromate, chromic acid, potassium permanganate, mercurials, picrates, formalin, paraformaldehyde, amine-reactive NHS-ester crosslinkers such as bis[sulfosuccinimidyl] suberate (BS3), etc. as evidenced by Van Der Oost (col 45, lines 16-33; and col 99, lines 48-65). Hong teaches that the targeted RNA-induced silencing complexes (RISCs) are then isolated from the crude lysate by immunoprecipitation by using anti-Argonaute antibody-conjugated magnetic beads, wherein the antibody captures the non-denatured (native) Argonaute proteins (interpreted as contacting an RNA sample with miRNA in the presence of Ago2 protein; interpreting IP as isolating RSIC as complexes; lysing prior to IP; using Ago2 antibody; and where non-chimeric molecules of interest are miRNA, claims 1, 3, 7, 8 and 10) (pg. 100, Section 1.6.1, first partial paragraph, lines 1-4). Hong teaches that after immunoprecipitation, sequential ligation of miRNA 5’-mismatch repairer and then miRNA 3’-mismatch repairer is performed because both ends of the miRNA might stick out from the Argonaute protein (interpreted as ligating miRNA molecules to the RNA molecules within the complex, claim 1) (pg. 100, Section 1.6.1, first partial paragraph, lines 4-6). Hong teaches that RNA digestion is performed to trim the RNAs protruding from the RISCs to the length sufficient for adaptor ligation (interpreting adaptor ligation as ligating miRNA molecules to the RNA molecules within the complex, claim 1) (pg. 100, Section 1.6.1, first partial paragraph, lines 9-10). Hong teaches that the Stem-Loop Adaptor (RNA) is used to connect the miRNAs and their corresponding target site to generate the chimeric sequences that represent the miRNA-target site interactions; and subsequently, the Argonaute protein is removed by digestion using proteinase K at 4℃, where this low temperature (4℃) keeps the RNA duplexes in duplex form (interpreting adaptor ligation as ligating miRNA molecules to the RNA molecules within the complex; and forming chimeric RNA molecules; and digesting Ago2 proteins before enrichment, claims 1 and 17) (pg. 100, Section 1.6.1, last partial paragraph, lines 6-10). Hong teaches that the chimeric sequence quantification assay uses a hydrolysis probe (Chimera Quanti Probe), and two primers (interpreted as a quantification assay, claim 1) (pg. 181, Section 4.11.3.1; first full paragraph). Hong teaches that the use of TargetScan, which was first developed prediction algorithm for predicting mammalian miRNA targets, wherein the algorithm seeks for seed matches at 3’ UTR of mRNA of the query organism such as humans, such that the definition of seed match is the full complementarity of miRNA positions 2 to 8 (the 7-mer seed region of miRNA) to anywhere in the 3’ UTR of candidate target mRNAs (interpreted as computationally identifying RNA molecules of interest, claim 1) (pg. 67, Section 1.4.1, first full paragraph), where it is known that 3’-UTR corresponds to mature mRNA of a gene including APP, ATG9A, BTG2 and ULK1 as evidenced by Fotin-Mleczek (col 13, lines 7-8 and 15-16; col 19, line 42; col 20, line 14; col 21, line 10; and col 48, line 47). Hong teaches a flow chart of RIINGS as shown in Figure 3.1 including: UV irradiation to crosslink the effecting Argonaute complex, which in vivo includes an Argonaute protein, a miRNA and a target RNA; ligation of Reattaching Adaptor and Stem-Loop Adaptor (RNA); a first mismatch-repairing step; a second mismatch-repairing step; generation of chimeric sequences by RT-PCR; and sequence analysis of the chimeric library by NGS (interpreted as converting RNA into libraries that can be subjected to sequencing, claim 9) (pg. 105; Figure 3.1). Figure 3.1 is shown below:
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Hong teaches that RNA duplexes are captured by Library Enrichment Beads (interpreted as enriching non-chimeric RNA and chimeric RNA, claim 1) (pg. 101, Section 1.6.1; first full paragraph, line 1). Hong teaches that a second round of ligating miRNA 5’- and 3’-mismatch repairers is performed at the 5’ and 3’ ends of miRNAs respectively and sequentially, such that these two repairers generate new ends on the mismatched ends and these newly generated ends are potentially able to match their corresponding target sequence; and the gap between the miRNA 5’ end and the Stem-Loop Adaptor (RNA) 3’end is then repaired (filled) by the ligation of some short fillers (RNA tetramers), such that during this ligation process, the 3’ end of the miRNAs is also extended, finally reaches and then passes the restriction enzyme cleavage site on the “Re-attaching Adaptor”; and after these ligation steps, the chimeric sequences are generated (pg. 101, Section 1.6.1; first full paragraph, lines 1-9). Hong teaches that the chimeric sequences can be eluted by the restriction enzyme BanI because the Reattaching Adaptor includes a BanI cutting site; and finally, the chimeras are recovered through reverse transcription, PCR and NGS (interpreted as enriching cDNA; PCR; and sequencing the PCR products, claims 1 and 15) (pg. 101, Section 1.6.1; last full paragraph, lines 1-3). Hong teaches that the stem-loop adaptor has to be ligated to the 3' end of the miRNA target as shown in the scheme of RIINGS (Figure 3.1), such that this adaptor effectively extends the 3' end of the miRNA target and flips it over and backward, and positions the 3' end of the “miRNA target” and the 5' end of the miRNA in a head-to-tail manner with a gap in between; and the loop of the adaptor conceals a probe-binding site because it is in fact the binding site of a specific probe, wherein this binding site facilitates the quantification of valid cDNA by means of qPCR or digital PCR in a library prepared by RIINGS (interpreted as enriching with probes, claim 1) (pg. 126, Section 4.6; last partial paragraph; and pg. 127, first partial paragraph). Hong teaches that the miRanda algorithm is also commonly used for predicting target sites of miRNAs; and uses criteria similar to those of TargetScan, and was initially built for predicting miRNA target sites in fly and humans, wherein it first aligns miRNAs (the whole sequence of each miRNA) to the 3’ UTR of target mRNAs to recognize highly complementary sequences (interpreted as computationally identifying RNA molecules of interest, claim 1) (pg. 68, Section 1.4.3, first partial paragraph). Hong teaches a method for the a rapid, specific and non-susceptible to proteinase K digestion for recovering released miRNA-target RNA duplex, where an adaptor called “Re-attaching Adaptor” is ligated to the 5’ end of the target RNA on the isolated AGO complex (Figure 3.1), wherein it is a DNA adaptor carrying (from 5’ end) a specific sequence, a restriction enzyme site and a few bases of RNA at its 3’ end, such that the specific sequence can pair up with an antisense DNA oligo conjugated on “Library Enrichment beads”, such that the “Library Enrichment beads” recover the miRNA-target RNA duplex rapidly and efficiently through annealing; and that cleavage of the restriction enzyme site by the corresponding restriction enzyme finally releases the chimeric sequence product from the “Library Enrichment beads” (Figure 3.1), wherein the released chimeric sequence is in a stem-loop structure with a sticky end at the end of the stem, such that this sticky end facilitates the ligation of the sequencing adaptor to the chimeric sequence (interpreted as performing an enrichment step; coupling biotinylated probes; hybridization probes; the probes are antisense probes; and eluting the enriched RNA molecules from the beads, claims 1, 51 and 52) (pg. 329, last full paragraph).
Regarding claim 9, Hong teaches a flow chart of RIINGS as shown in Figure 3.1 including:
UV irradiation to crosslink the effecting Argonaute complex, which in vivo includes an Argonaute protein, a miRNA and a target RNA; ligation of Reattaching Adaptor and Stem-Loop Adaptor (RNA); a first mismatch-repairing step; a second mismatch-repairing step; generation of chimeric sequences by RT-PCR; and sequence analysis of the chimeric library by NGS (interpreted as converting RNA into libraries that can be subjected to sequencing, claim 9) (pg. 105; Figure 3.1).
Regarding claims 12 and 16, Hong teaches that the hydrolysis probe has the structure of Figure 4.11.3, which comprises miRNA, target RNA target site, stem-loop adaptor and sequencing adaptors (interpreted as comprising RNA; and 100% complementary to miRNA, claims 12 and 16) (pg. 180, Figure 4.11.3). Figure 4.11.3 is shown below:
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Regarding claim 13, Hong teaches that the use of TargetScan, which was first developed prediction algorithm for predicting mammalian miRNA targets, wherein the algorithm seeks for seed matches at 3’ UTR of mRNA of the query organism such as humans, such that the definition of seed match is the full complementarity of miRNA positions 2 to 8 (the 7-mer seed region of miRNA) to anywhere in the 3’ UTR of candidate target mRNAs (interpreted as mapping to specific genes or 3’-UTR of genes, claim 13) (pg. 67, Section 1.4.1, first full paragraph). Hong teaches that the miRanda algorithm is also commonly used for predicting target sites of miRNAs; and uses criteria similar to those of TargetScan, and was initially built for predicting miRNA target sites in fly and humans, wherein it first aligns miRNAs (the whole sequence of each miRNA) to the 3’ UTR of target mRNAs to recognize highly complementary sequences (interpreted as mapping to specific genes or 3’-UTR of genes, claim 13) (pg. 68, Section 1.4.3, first partial paragraph).
Regarding claim 19, Hong teaches that unlike CLASH, Mapping RNA interactome in vivo (MARIO) is a method that requires the insertion of a biotinylated short linker between the two interacting RNAs in RNA chimera construction, such that the chimeras can thus be enriched from the sample by this biotinylated short linker (Figure 1.5.5), wherein the chimera enrichment is a very distinctive advantage of MARIO (interpreted as enrichment increases the proportion of chimeric reads in the library, claim 19) (pg. 88, Section 1.5.6, first full paragraph).
Regarding claims 51 and 52, Hong teaches that the chimeras can be enriched from the sample by using a biotinylated short linker (interpreted as a biotinylated probe, claim 52) (pg. 88, Section 1.5.6, first full paragraph). Hong teaches that with the intention of chimera enrichment, a DNA oligo complementary to the biotinylated RNA linker is added to the purified RNAs, such that this DNA oligo forms a duplex with the RNA linker, wherein T7 exonuclease (an exonuclease with RNase H activity) is added to the mixture to remove all the end-located biotinylated RNA linkers, but not the linkers located internally, such that all the chimeras can be easily enriched by streptavidin beads (interpreted as two or more miRNA molecules; and streptavidin beads, claim 52) (pg. 88, last partial paragraph; and pg. 89, first partial paragraph). Hong teaches a method for the rapid, specific and non-susceptible to proteinase K digestion for recovering released miRNA-target RNA duplex, where an adaptor called “Re-attaching Adaptor” is ligated to the 5’ end of the target RNA on the isolated AGO complex (Figure 3.1), wherein it is a DNA adaptor carrying (from 5’ end) a specific sequence, a restriction enzyme site and a few bases of RNA at its 3’ end, such that the specific sequence can pair up with an antisense DNA oligo conjugated on “Library Enrichment beads”, such that the “Library Enrichment beads” recover the miRNA-target RNA duplex rapidly and efficiently through annealing; and that cleavage of the restriction enzyme site by the corresponding restriction enzyme finally releases the chimeric sequence product from the “Library Enrichment beads” (Figure 3.1), wherein the released chimeric sequence is in a stem-loop structure with a sticky end at the end of the stem, such that this sticky end facilitates the ligation of the sequencing adaptor to the chimeric sequence (interpreted as performing an enrichment step; coupling biotinylated probes; hybridization probes; the probes are antisense probes; and eluting the enriched RNA molecules from the beads, claims 1, 51 and 52) (pg. 329, last full paragraph).
Hong does not specifically exemplify crosslinking agents as recited in claim 5 (claim 5); antisense nucleic acid probes having a length between 10 bp and 100 bp (claim 11); antisense nucleic acid probes having a length between 10 bp and 5000 bp (claim 14); where the enrichment step produces about 5% to 30% chimeric reads out of all uniquely mapped reads (claim 18); where the overall chimeric read population is increased 20-fold (claim 20); and where the Ago2 protein includes a gene selected from APP, ATG9A, BET2 and ULK1 (claim 25).
Regarding claims 5, 11, 14, 18, 20 and 25, Xu teaches high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (CLIP), wherein the isolated Argonaute protein (Ago) binding sites are analyzed computationally to infer the possible microRNA(s) that could have been responsible for tethering Ago to those binding sites (pg. 259, col 2, first partial paragraph, lines 4-9). Xu teaches that a crosslinking and ligation approach termed CLASH was reported in which crosslinked miRNA-target sequence are ligated to generate chimeras for sequencing, wherein the method should overcome the ambiguity in assigning the Ago-bound sequence to the microRNA responsible for recruiting the target gene to the RISC complex, where a drawback of the method is its relatively low efficiency of chimera generation and capture (pg. 259, col 2, last partial paragraph, lines 14-20; and pg. 260, col 1, first partial paragraph, line 1). Xu teaches that TargetLink is used for identifying target genes directly bound by a specific microRNA, wherein TargetLink involves crosslinking microRNA and its target genes in intact live cells by UV illumination, and cell lysates containing the crosslinked microRNA-Argonaute-mRNA ternary complexes were prepared and subjected to affinity purification using locked nucleic acid (LNA) as the capture probe to pull down complementary microRNA and its crosslinked target genes (interpreted as anti-sense nucleic acid probes; pull down; LNA; inherently produces 5% to 30% chimeric reads; and inherently increases reads by at least 20-fold, claims 11, 14, 18 and 20) (pg. 260, col 1; second full paragraph, lines 1-8). Xu teaches that TargetLink is based on the principles of RNA photo-crosslinking and antisense purification of crosslinked RNA complexes, wherein because microRNAs are only ~22 nt long, locked nucleic acid (LNA) was applied as the affinity probe to pull down microRNA and associated protein complexes, wherein LNA oligonucleotides bind to complementary RNA with very high affinity and specificity to yield remarkably stable LNA/RNA duplexes, and antisense LNA oligonucleotides as short as 8-mer have been successfully used to knock down miRNA (interpreted as anti-sense nucleic acid probes between 10-100 or 10-5000 bp; and LNA, claims 11 and 14) (pg. 260, col 2; first full paragraph, lines 1-9). Xu teaches that the resulting file containing the unique mapped reads reflected the distribution of affinity purified RNA fragments across the transcriptome, and it was used for detecting the enrichment of sequences resulting from LNA affinity purification (interpreted as increases in chimeric reads, claims 18 and 20) (pg. 261, col 1, last partial paragraph; and pg. 262, col 1, first partial paragraph). Xu teaches that the LNA affinity probe selectively pulled down more transcripts in UV crosslinked samples through the crosslinked ternary complexes containing miR-21/Ago/targets (pg. 262, col 2, first partial paragraph).
Although the combined references of Hong and Xu do not specifically teach that crosslink agents include formalin, formaldehyde, etc., Hong does teach that crosslinking can be achieved by 254 nm UV light or 365 nm UV light with the assistance of 4-thiouridine (4SU), where it is known in the art that cells or complexes can be fixed or crosslinked using suitable fixatives and crosslinkers including: formaldehyde, glutaraldehyde, ethanol-based fixatives, methanol-based fixatives, acetone, acetic acid, osmium tetraoxide, potassium dichromate, chromic acid, potassium permanganate, mercurials, picrates, formalin, paraformaldehyde, amine-reactive NHS-ester crosslinkers such as bis[sulfosuccinimidyl] suberate (BS3), etc. as evidenced by Van Der Oost; and Xu does teach high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (CLIP); a crosslinking and ligation approach (CLASH); and crosslinking microRNA and its target genes in intact live cells by UV illumination, such that one of ordinary skill in the art would clearly recognize that a variety of crosslinking agents can be used in a crosslinking reaction including the chemical agents such as formalin, formaldehyde, etc.
Although the combined references of Hong and Xu do not specifically exemplify Ago2 includes a gene selected from APP, ATG9A, BTG2 and ULK1, Hong does teach that the use of TargetScan, which was first developed prediction algorithm for predicting mammalian miRNA targets, wherein the algorithm seeks for seed matches at 3’ UTR of mRNA of the query organism such as humans, such that the definition of seed match is the full complementarity of miRNA positions 2 to 8 (the 7-mer seed region of miRNA) to anywhere in the 3’ UTR of candidate target mRNAs, where it is known that 3’-UTR corresponds to mature mRNA of a gene including APP, ATG9A, BTG2 and ULK1 as evidenced by Fotin-Mleczek; and Xu does teach reads mapped to a candidate miR-21 target gene MKNK2; and the identification of miR-21 target genes using deep sequencing data, such that one of ordinary skill in the art would clearly recognize that any specific gene of interest including the 3’ UTR of any candidate target mRNAs can be selected and/or investigated using the methods taught supra including for the identification of miR target genes bound to specific miRNA such as APP, ATG9A, BTG2 and ULK1.
“It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from there having been individually taught in the prior art.” In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980). Moreover, it is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of identifying target genes directly bound by a specific microRNA as exemplified by Xu, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of identifying the entire interactome of microRNA in a specific cell type or tissue using the RINGS method as disclosed by Hong to include the TargetLink method of antisense purification of crosslinked RNA complexes using antisense nucleic acids as taught by Xu with a reasonable expectation of success in efficiently identifying the target set of a specific microRNA in intact cells; in enhancing the generation of microRNA-target RNA chimeras from all interacting pairs during library preparation; and/or in obtaining a file of uniquely mapped reads reflecting the distribution of affinity purified RNA fragments across the transcriptome.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly
rejected under 35 USC §103(a) as obvious over the art.
Response to Arguments
Applicants’ arguments filed July 13, 2026 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) neither Hong nor Xu, whether considered individually or in combination, teaches or suggests this particular combination of steps, and the Office Action fails to articulate any reasoned basis, supported by a reasonable expectation of success, for modifying Hong and Xu to arrive at the amended claimed methods including that Xu’s Target Link operates on a fundamentally different principle and uses a different method (Applicant Remarks, pg. 13, last partial paragraph through pg. 14, second full paragraph); (b) the modification of Hong with Xu would render the prior art method unsuitable for the intended purpose (Applicant Remarks, pg. 14, last full paragraph, lines 1-8); (c) Xu teaches away from chimera-ligation approaches by identifying methods such as CLASH as having a “drawback” and “relatively low efficiency” (Applicant Remarks, pg. 14, last full paragraph, lines 8-13); (d)
The Examiner offers no evidence that Xu's TargetLink "inherently" produces about 5% to 30% chimeric reads (Claim 18) and inherently increases the overall chimeric read population by at least 20-fold (Claim 20) (Applicant Remarks, pg. 15, first full paragraph); and (e) claim 25 is not obvious with respect to the genes of interest and because the combination does not render claim 1 obvious, it does not render claim 25 obvious (Applicant Remarks, pg. 15, first full paragraph).
Regarding (a) and (b), Applicant’s assertion that neither Hong nor Xu, whether considered individually or in combination, teaches or suggests this particular combination of steps, and the Office Action fails to articulate any reasoned basis, supported by a reasonable expectation of success, for modifying Hong and Xu to arrive at the amended claimed methods including that Xu’s Target Link operates on a fundamentally different principle and uses a different method; and the modification of Hong with Xu would render the prior art method unsuitable for the intended purpose, is not found persuasive. Applicant merely recited the steps as recited in instant claim 1 without indicating what specific steps Applicant believes are not taught by the combined references. Applicant did not distinctly and specifically point out the supposed errors in the Examiner’s action as required by 37 CFR 1.111(b). Thus, the claims remain rejected for the reasons already of record.
Regarding an articulated reason to modify Hong and Xu, MPEP 2144(I) states:
The rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art, established scientific principles, or legal precedent established by prior case law. In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988); In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992). See also In re Kotzab, 217 F.3d 1365, 1370, 55 USPQ2d 1313, 1317 (Fed. Cir. 2000) (setting forth test for implicit teachings); In re Eli Lilly & Co., 902 F.2d 943, 14 USPQ2d 1741 (Fed. Cir. 1990) (discussion of reliance on legal precedent); In re Nilssen, 851 F.2d 1401, 1403, 7 USPQ2d 1500, 1502 (Fed. Cir. 1988) (references do not have to explicitly suggest combining teachings); Ex parte Clapp, 227 USPQ 972 (Bd. Pat. App. & Inter. 1985) (examiner must present convincing line of reasoning supporting rejection); and Ex parte Levengood, 28 USPQ2d 1300 (Bd. Pat. App. & Inter. 1993) (reliance on logic and sound scientific reasoning).
Moreover, Applicants are reminded that the motivation for combining the teachings of the prior art may be different from Applicants’ motivation to make the disclosed compositions. The fact that applicant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). The Office has provided motivation for modifying the method for identifying the interactome of miRNA using the methods taught by Hong to include the antisense purification of crosslinked RNA complexes as taught by Xu with a reasonable expectation of success in efficiently identifying the target set of a specific microRNA in intact cells; and in enhancing the generation of microRNA-target RNA chimeras from all interacting pairs during library preparation.
Applicant states that TargetLink operates on a fundamentally different principle, but it is unclear what process Applicant is comparing TargetLink to, and whether it operates on a fundamentally different principle as compared to Hong, Xu, and/or to the instant invention. The Examiner has assumed, arguendo, that Applicant is arguing that Hong and Xu are non-analogous art; and Xu would render Hong unfit for its intended purpose. To that end, MPEP 1504.03(II)(A)(2) states that:
When modifying the surface of a primary reference so as to provide it with an attractive appearance, it is immaterial whether the secondary reference is analogous art, since the modification does not involve a change in configuration or structure and would not have destroyed the characteristics (appearance and function) of the primary reference. See In re Glavas, 230 F.2d 447, 109 USPQ 50 (CCPA 1956).
The Examiner notes that:
Hong and Xu are analogous art. Hong is directed to a method for identifying miRNA-RNA interactome in cells and miRNA-Ago2-RNA complexes, while Xu is directed to a method for identifying the endogenous target set of specific miRNA in intact living cells including computationally determining the accessibility of RNAs in the miRNA-Ago complex.
The Xu reference is cited for reading on the dependent claims (e.g., it does not read on the method recited in instant claim 1) (e.g., the length of antisense probes, crosslinking reagents, computational methods, etc.).
The teachings of Xu do not involve a change in configuration or structure of the method disclosed by Hong, and it would not have destroyed the characteristics (appearance and function) of the primary reference.
Adding the computational method of Xu to the methods taught by Hong would not make Hong unsuitable for its intended purpose.
Thus, the claims remain rejected.
Regarding (c), it is noted that disclosed examples and preferred embodiments do not constitute a teaching away from a broader disclosure or nonpreferred embodiments. In re Susi, 440 F.2d 442, 169 USPQ 423 (CCPA 1971). Moreover,
"The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill the art, including nonpreferred embodiments (underline added). See In re Heck, 699 F.2d 1331, 1332-33,216 USPQ 1038, 1039 (Fed. Cir. 1983); In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275,277 (CCPA 1968); Merck & Co. v. Biocraft Laboratories, 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); and Upsher-Smith Labs. v. Pamlab, LLC, 412 F.3d 1319, 1323, 75 USPQ2d 1213, 1215 (Fed. Cir. 2005).
Additionally, a reference teaches away “when a person of ordinary skill, upon reading the reference, would be discouraged from following the path set out in the reference, or would be led in a direction divergent from the path that was taken” in the claim. Galderma Labs., L.P. v. Tolmar, Inc., 737 F.3d 731, 738 (Fed. Cir. 2013). A reference that “merely expresses a general preference for an alternative invention but does not criticize, discredit, or otherwise discourage investigation into” the claimed invention does not teach away. Id. Furthermore, MPEP 2141.02(VI) states that "the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed…." In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004). Applicant’s assertion that Xu teaches away from chimera-ligation approaches by identifying methods such as CLASH as having a “drawback” and “relatively low efficiency”, is not found persuasive. Again, Xu reads on the dependent claims, such that Xu is not cited to support the limitations as recited in instant claim 1. Moreover, Xu clearly does not criticize, discredit, or otherwise discourage investigation into CLASH. Xu’s statement that a drawback to CLASH is its “relatively low efficiency of chimera generation and capture” hardly rises to the level of “teaching away” from the RIINGS method, TargetLink, crosslinked miRNA-target sequence, identifying native targets of endogenous miRNAs, or any other method disclosed within the combined references. Thus, the claims remain rejected.
Regarding (d), as noted in MPEP § 2112.01(I), where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). Moreover, MPEP § 2112.02(I) states that under the principles of inherency, if a prior art device, in its normal and usual operation, would necessarily perform the method claimed, then the method claimed will be considered to be anticipated by the prior art device. When the prior art device is the same as a device described in the specification for carrying out the claimed method, it can be assumed the device will inherently perform the claimed process. In re King, 801 F.2d 1324, 231 USPQ 136 (Fed. Cir. 1986).
Applicants’ assertion that The Examiner offers no evidence that Xu's TargetLink "inherently" produces about 5% to 30% chimeric reads (Claim 18) and inherently increases the overall chimeric read population by at least 20-fold (Claim 20), is not found persuasive. As an initial matter, the Examiner notes that instant claims 18 and 20 are dependent claims. Furthermore, please see the 35 USC 112(b) rejection supra, wherein instant claims 18 and 20 are indefinite for the recitation of the amount of increase in chimeric reads or an overall chimeric read population because reads are not recited to be obtained or present in instant claim 1. Additionally, the enrichment step as recited in claim 1 merely requires contacting PCR products with miRNA-specific hybridization probes, such that this method of enrichment must therefore inherently provide the recited quantity of chimeric reads, and/or the recited increase in overall chimeric read population. Hong clearly teaches the enrichment step as recited in claim 1 and, therefore, must obtain the recited read populations and/or the increase in proportion of chimeric reads. Thus, the claims remain rejected.
Regarding (e), Applicants’ assertion that claim 25 is not obvious with respect to the genes of interest; and because the combination does not render claim 1 obvious, it does not render claim 25 obvious, is not found persuasive. Please see the Examiner’s response to Applicant’s arguments discussed supra. It is noted that the gene of interest are recited in instant dependent claim 25. In the rejection supra, the Examiner has provided reasoning why the target RNA molecule of chimeric RNA molecules can be a transcript selected from one of the recited genes. Thus, the claims remain rejected.
Conclusion
Claims 1, 3-20, 25, 51 and 52 remain rejected.
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 AMY M BUNKER whose telephone number is (313) 446-4833. The examiner can normally be reached on Monday-Friday (6am-2:30pm).
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/AMY M BUNKER/Primary Examiner, Art Unit 1684
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