DETAILED ACTION
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 .
Office Action: Notice
Any objection or rejection of record in the previous Office Action, mailed 9/11/2025, which is not addressed in this action has been withdrawn in light of Applicants' amendments and/or arguments. This action is FINAL.
Election/Restrictions
Applicant’s election without traverse of Group I in the reply filed on June 12, 2025 is acknowledged. Claims 22-24 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected Groups 2 and 3, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 6/12/2025. Thus, claims 1-3, 5-11, 13-14 and 15-20 are under examination (6/12/2025).
The original characterization of the elected Group 1 claims, as well as the scope of claim 22, is upheld, although the Applicant’s assertion is noted. The Applicant asserts that claim 1 is broader than merely purifying adapter-ligated DNA or binding using PEG/salt concentrations, however the claim language in its broadest reasonable interpretation recites a poly (alkylene oxide) polymer and a salt for binding nucleic acids, which squarely corresponds to the methods described in the restriction/election requirement (6/12/2025). Similarly, while the Applicant argues that claim 22 only optionally recites fragmentation and repair, the instant claim nevertheless encompasses such subject matter (6/12/2025). Therefore, the grouping was properly made to ensure distinct inventions were not examined together.
Claim Status
Claims 1-3, 5-11, 13-20 and 25-26 are under examination (3/11/2026). Claims 2, 5-10, 13-18 and 20 have been amended (3/11/2026). Claims 25-26 are new (3/11/2026). No new matter was added.
Priority
Claims 1-3, 5-11, 13-14 and 15-20 and 25-26 receive a priority date of 6/28/2019, the effective filing date of European Provisional Patent EP19183295.5.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure
statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information
submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be
incorporated into the specification but must be submitted in a separate paper." Therefore, unless
the references have been cited by the examiner on form PTO-892, they have not been
considered.
The information disclosure statements (IDS) submitted on 3/11/2026 and 4/23/2026 are being considered by the examiner.
Objections Withdrawn
Specification:
The objections to the specification due to the use of a trademark or tradenames are withdrawn in view of Applicant’s amendments.
Claims
The objections to claims 9 and 18 for minor informalities (i.e., spacing issues) are withdrawn in view of Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 112(b)
The rejections of claims 1-3, 5-11, 13-14 and 15-20 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, are withdrawn in view of Applicant’s arguments and subsequent amendments of claims 2, 5-10, 13-18 and 20, to address antecedent basis and indefinite issues.
Claim Rejections – 35 USC § 102
The 102 (a) (1) and 102 (a) (2) rejections of claims 2, 5-11, 13-14 and 15-20 are withdrawn in view of Applicant’s arguments and substantial amendments (3/11/2026). Specifically, newly amended dependent claims include limitations that are not expressly disclosed by O’Neil, including “size cut-off values”, specific PEF and salt concentration ranges, adapter-ligated DNA enrichment, and other narrowed process parameters recited in these claims.
Double-Patenting
The rejection of claims 1-3, 5-11, 13-14 and 15-20, on the ground of obviousness-type nonstatutory double patenting as being unpatentable over claims 1-3, 6-7, 9-14, 16-18 and 22-24 of U.S. patent 8,729,252 is withdrawn in view of Applicant’s arguments, specifically the distinct focus of the presently claimed size-selective enrichment methodology as compared to the silica magnetic particle clustering concerns addressed in the reference application.
Rejections Maintained
Claim Rejections – 35 USC § 102
Claims 1 and 3 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by O’Neill et al., (WO 2018/144854 A1, published 8/9/2018).
Regarding claim 1, O’Neill teaches a composition comprising such isolated oligonucleotide or plurality of oligonucleotides (Paragraph 23, lines 1-2). Further, O’Neill teaches that the isolation or size selection methodology can incorporate modifications comprising a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., polyalkylene glycol, such as polyethylene glycol) (Figures 3-4; Paragraph 56, lines 1-10). Further, O’Neill teaches a binding agent (702), here an aptamer for purposes of illustration, tethered to a substrate (701) where the binding agent (702) can be covalently attached to substrate (701) or alternatively the binding agent (702) may also be non-covalently attached (i.e., binding agent (702) can comprise a label which can be attracted to the substrate, such as a biotin group which can form a complex with an avidin/streptavidin molecule that is covalently attached to the substrate (Figure 7; Paragraph 60, lines 1-5). Specifically, O’Neill teaches that the pH and/or ionic strength of the solution can be adjusted with various acids, bases, buffers or salts, including without limitation sodium chloride (NaCl), phosphate-buffered saline (PBS), tris-buffered saline (TBS), sodium phosphate, potassium chloride, potassium phosphate, sodium citrate and saline-sodium citrate (SSC) buffer (Paragraph 236, lines 1-10).
Further, O’Neill teaches that the previously described isolation or size selection method can be used with an antibody in order to isolate a vesicle that can be bound to a solid substrate such as a well, such as commercially available plates where each well can be coated with the antibody and the antibody is used to isolate a vesicle bound to a solid substrate such as an array (Paragraph 264, lines 1-5).
Additionally, O’Neill teaches that the targeted or bound oligos or antibodies can be eluted, captured via their biotin linkage and then combined again with normal biological sample where the unbound oligos are then added again to disease-derived biological sample and isolated (Figure 4, Paragraph 57, lines 1-5). O’Neill also teaches that these capture arrays can be customized into vesicles that can be isolated from a biological sample using a system that uses multiple antibodies that are specific to the most abundant proteins found in a biological sample, such as blood and that such a system can remove up to several proteins at once, thus unveiling the lower abundance species such as cell-of-origin specific vesicles (Paragraph 225, lines 1-5).
Regarding claim 3, O’Neill teaches that the previously described isolation or size selection method includes removal of highly abundant proteins and other non-desired entities that can further be facilitated with a non-stringent size exclusion step, where the sample can be processed using a high molecular weight cutoff size exclusion step to preferentially enrich high molecular weight vesicles apart from lower molecular weight proteins and other entities (Paragraph 223, lines 1-5). Further, O’Neill teaches that if a vesicle population is detected or isolated using two, three, four or more binding agents in a differential detection or isolation of a vesicle from a heterogeneous population of vesicles, the particular binding agent profile for the vesicle population provides a biosignature for the particular vesicle population (Paragraph 253, lines 15-20).
Further, O’Neill teaches that the previously described array can incorporate capture agents that can also be attached to beads that can be manipulated to move through the microfluidic channels and are attached to magnetic beads (Paragraph 184, lines 5-10).
O’Neill also teaches that one or more reagents can be the one or more aptamer, a buffer, blocker, enzyme, or combination thereof, and the one or more reagents may comprise any useful reagents for carrying out the subject methods, including without limitation aptamer libraries, substrates such as microbeads or planar arrays or wells, reagents for biomarker and/or microvesicle isolation (i.e., via chromatography, filtration, ultrafiltration, centrifugation, ultracentrifugation, flow cytometry, affinity capture (i.e., to a planar surface, column or bead), polymer precipitation, and/or using microfluidics), aptamers directed to specific targets, aptamer pools that facilitate detection of a biomarker/microvesicle population, reagents such as primers for nucleic acid sequencing or amplification, arrays for nucleic acid hybridization, detectable labels, solvents or buffers and the like, various linkers, various assay components, blockers, and the like (Paragraph 622, lines 1-10). Also, O’Neill teaches that aptamers were then pooled after ligating barcodes and adapter sequences (Paragraph 644, lines 1-2). O’Neill teaches that the previously described method can be applied to create a sequencing library via the Illumina TruSeq Stranded mRNA HT Sample Prep and subjected to HiSeq Rapid paired-end 100 bp plus sequencing on an Illumina HiSeq 2500 Ultra-High- Throughput Sequencing System (Paragraph 1280, lines 5-10).
O’Neill teaches each and every limitation of claims 1 and 3, and therefore O’Neill anticipates claims 1 and 3.
Applicant’s Response: The Applicant argues that O’Neill has been improperly characterized by the Office because O’Neill is directed to aptamer selection, biomarker detection, and extracellular vesicle isolation rather than a poly(alkylene oxide) polymer-based size-selective nucleic acid enrichment method. Applicant further contends that the cited portions of O’Neill do not disclose the claimed size-selective enrichment process, including the recited cut-off values, selective binding and elution of nucleic acids based on size, adapter-ligated DNA enrichment, and the specific PEG/salt conditions recited in the amended claims.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are found partially persuasive, as discussed below, as the existing rejection was withdrawn for all dependent claims with the expectation of claim 3.
However, Applicant’s arguments are not persuasive with respect to unamended independent claim 1. While Applicant contends that O’Neil is directed to aptamer selection, biomarker detection, and extracellular vesicle isolation rather than a poly (alkylene oxide) polymer-based size-selective nucleic acid enrichment method, a prior art reference is available for all that it reasonably teaches and is not limited to its primary stated purpose (see MPEP 2121). Further, during examination, claims are given their broadest reasonable interpretation consistent with the specification (see MPEP 2111).
Under its broadest reasonable interpretation, claim 1 broadly recites a method comprising; (i) preparing a binding mixture comprising a nucleic acid sample, a poly (alkylene oxide) polymer, and a salt; (ii) binding nucleic acid molecules to a solid phase comprising a functional group; (iii) contacting the solid phase with a reagent composition comprising a poly(alkylene oxide) polymer and a salt; (iv) selectively eluting non-target nucleic acid molecules. Claim 1 does not recite any particular nucleic acid type, adapter-ligated DNA, sequencing library preparation, PEG molecular weight, PEG concentration range, salt concentration range, magnetic particle composition, fragment size threshold, or cut-off value. Rather, claim 1 broadly encompasses nucleic acid isolation and size-selection methodologies employing poly (alkylene oxide) polymers, salts, and solid-phase separation techniques.
The rejection of claim 1 is based on the specific disclosures of O’Neill regarding nucleic acid isolation and size selection methodologies, including the use of poly (alkylene oxide) compounds such as polyethylene glycol, salts and buffer systems for controlling binding and elution conditions, solid-phase capture and separation techniques, and recovery of nucleic acid molecules, as shown in the above rejection. The Applicant’s arguments focus primarily on the intended purpose and broader context of O’Neill rather than the teachings actually relied upon in the rejection. While Applicant’s amendments to dependent claims, with the exception of dependent claim 3, introduce additional limitations directed to specific size cut-off values, PEG and salt concentration ranges, adapter-ligated DNA enrichment, and other narrowed process parameters not expressly disclosed by O’Neill, those limitations are not recited in independent claim 1.
Accordingly, the rejection of claims 1 and 3 is maintained under 35 USC 102(a)(1) and 102(a)(2).
Double Patenting
Claims 1, 3 and 6-11 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5-12, and 25-26 of co-pending U.S. patent application 17/620,612.
The amended claims received October 21, 2022 are cited in this non-statutory double patenting rejection.
Claim 1 of ‘615 claims a poly (alkylene oxide) polymer-based size selective nucleic acid
enrichment method for enriching target nucleic acid molecules from a nucleic acid containing
sample which comprises target nucleic acid molecules and non-target nucleic acid molecules,
wherein the target nucleic acid molecules are longer than the non-target nucleic acid molecules,
the method comprising:
(a) preparing a binding mixture comprising the nucleic acid containing sample,
a poly (alkylene oxide) polymer and a salt and binding nucleic acid molecules to a solid phase which comprises a functional group, wherein the bound nucleic acid molecules comprise
target nucleic acid molecules;
(b) optionally separating the solid phase with the bound nucleic acid molecules
from the remaining sample;
(c) contacting the solid phase with the bound nucleic acid molecules at least once with a reagent composition comprising a poly (alkylene oxide) polymer and a salt to selectively elute non-target nucleic acid molecules, wherein the concentration (w/v) of the poly (alkylene oxide) polymer in the reagent composition of step (c) is lower than the concentration (w/v) of the poly (alkylene oxide) polymer in the binding mixture of step (a);
(d) optionally washing the bound target nucleic acid molecules; and
(e) eluting the bound target nucleic acid molecules from the solid phase.
Claim 1 of the ‘612 application recites a method with the same overall steps of binding nucleic acids in the presence of PEG and salt to a solid phase, separating, contacting with PEG/salt elution solution to establish a cut-ff, and selectively eluting nucleic acids below the cut-off. The differences in explicit concentration comparisons in the instant claim (i.e., PEG concentration in step (c) lower than in step (a)) represent only obvious variations of the method already recited in ‘612 claim 1.
In addition, step (d) of ‘612 is obvious over claim 1 of the instant application requiring a method of enriching target nucleic acid molecules from a nucleic acid containing sample which comprises target nucleic acid molecules and non-target nucleic acid molecules. Because step (c) of the instant application requires selectively eluting non-target nucleic acid molecules, it would have been obvious to separate this elution from the solid-phase based on the method of the instant application.
Claim 3 of the instant application depends from claim 1 and recites that the solid phase comprises acidic groups, carboxyl groups, or is provided by magnetic particles, or carboxylated magnetic particles (which is rendered obvious by claim 6 of ‘612).
Claim 6 of the instant application depends from claim 1 and recites that the poly (alkylene oxide) polymer is a polyethylene glycol, rendering obvious (which is rendered obvious by claim 7 of ‘612).
Claim 7 of the instant application depends from claim 1 and recites that the salt is a non-chaotropic salt, monovalent salt, alkali metal salt, or is sodium chloride (which is rendered obvious by claim 8 of ‘612).
Claim 8 of the instant application depends from claim 1 and requires wherein step (a) comprises adding a binding reagent to the nucleic acid containing sample to prepare the binding mixture, wherein the binding reagent comprises the poly (alkylene oxide) polymer, optionally a polyethylene glycol, and the salt (which is rendered obvious by claim 9 of ‘612).
Claim 9 of ‘615 depends from claim 8 and requires the binding conditions are exclusively established by contacting the binding reagent with the nucleic acid containing sample (which is rendered obvious by claim 10 of ‘612).
Claim 10 of ‘615 depends from claim 1 and recites that the reagent composition of (c) has the characteristic of (a) it comprises the poly (alkylene oxide) polymer, which is optionally a polyethylene glycol, in a concentration of at least 5% (w/v) (which is rendered obvious by claim 11 of ‘612).
Claim 11 of ‘615 depends from claim 8 and recites (c) the method comprises providing the reagent composition of step (c) by mixing the binding reagent with a dilution solution (which is rendered obvious by claim 12 of ‘612).
Accordingly, instant claims 1, 3 and 6-11 are not patentably distinct from claims 1, 5-12, and 25-26 of co-pending US Application No., 17/620,612. The dependent claims of the instant application recite only further optimizations of PEG concentration, molecular weight, salt type, or kit configurations, all of which are explicitly disclosed of obvious variations of the ‘612 claims.
Examiner’s Response to Traversal: Applicant’s request that the provisional nonstatuatory double patenting rejection be held in abeyance has been considered. The copending application remains pending and provisional rejection is therefore maintained pending final resolution of the claims in the copending application
New Rejections
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Claims 2, 5-11, 13-14 and 15-20 and 25-26 are rejected under 35 U.S.C. 103 as being unpatentable over O’Neill et al., (WO 2018/144854 A1, published 8/9/2018), as applied to claims 1 and 3 above, in view of Sauer et al. (“Sequential CaCl2, polyethylene glycol precipitation for RNase-free plasmid DNA isolation”, Analytical Biochemistry, 2008) and in further view of Shan et al. (“Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process”, Analytical Biochemistry, 2011).
As previously shown, O’Neill teaches a composition comprising such isolated oligonucleotide or plurality of oligonucleotides (Paragraph 23, lines 1-2). Further, O’Neill teaches that the isolation or size selection methodology can incorporate modifications comprising a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., polyalkylene glycol, such as polyethylene glycol) (Figures 3-4; Paragraph 56, lines 1-10). Further, O’Neill teaches a binding agent (702), here an aptamer for purposes of illustration, tethered to a substrate (701) where the binding agent (702) can be covalently attached to substrate (701) or alternatively the binding agent (702) may also be non-covalently attached (i.e., binding agent (702) can comprise a label which can be attracted to the substrate, such as a biotin group which can form a complex with an avidin/streptavidin molecule that is covalently attached to the substrate (Figure 7; Paragraph 60, lines 1-5). Specifically, O’Neill teaches that the pH and/or ionic strength of the solution can be adjusted with various acids, bases, buffers or salts, including without limitation sodium chloride (NaCl), phosphate-buffered saline (PBS), tris-buffered saline (TBS), sodium phosphate, potassium chloride, potassium phosphate, sodium citrate and saline-sodium citrate (SSC) buffer (Paragraph 236, lines 1-10).
Further, O’Neill teaches that the previously described isolation or size selection method can be used with an antibody in order to isolate a vesicle that can be bound to a solid substrate such as a well, such as commercially available plates where each well can be coated with the antibody and the antibody is used to isolate a vesicle bound to a solid substrate such as an array (Paragraph 264, lines 1-5).
Additionally, O’Neill teaches that the targeted or bound oligos or antibodies can be eluted, captured via their biotin linkage and then combined again with normal biological sample where the unbound oligos are then added again to disease-derived biological sample and isolated (Figure 4, Paragraph 57, lines 1-5). O’Neill also teaches that these capture arrays can be customized into vesicles that can be isolated from a biological sample using a system that uses multiple antibodies that are specific to the most abundant proteins found in a biological sample, such as blood and that such a system can remove up to several proteins at once, thus unveiling the lower abundance species such as cell-of-origin specific vesicles (Paragraph 225, lines 1-5).
Regarding claims 2-3 and 5, O’Neill teaches that the previously described isolation or size selection method includes removal of highly abundant proteins and other non-desired entities that can further be facilitated with a non-stringent size exclusion step, where the sample can be processed using a high molecular weight cutoff size exclusion step to preferentially enrich high molecular weight vesicles apart from lower molecular weight proteins and other entities (Paragraph 223, lines 1-5). Further, O’Neill teaches that if a vesicle population is detected or isolated using two, three, four or more binding agents in a differential detection or isolation of a vesicle from a heterogeneous population of vesicles, the particular binding agent profile for the vesicle population provides a biosignature for the particular vesicle population (Paragraph 253, lines 15-20).
Further, O’Neill teaches that the previously described array can incorporate capture agents that can also be attached to beads that can be manipulated to move through the microfluidic channels and are attached to magnetic beads (Paragraph 184, lines 5-10).
O’Neill also teaches that one or more reagents can be the one or more aptamer, a buffer, blocker, enzyme, or combination thereof, and the one or more reagents may comprise any useful reagents for carrying out the subject methods, including without limitation aptamer libraries, substrates such as microbeads or planar arrays or wells, reagents for biomarker and/or microvesicle isolation (i.e., via chromatography, filtration, ultrafiltration, centrifugation, ultracentrifugation, flow cytometry, affinity capture (i.e., to a planar surface, column or bead), polymer precipitation, and/or using microfluidics), aptamers directed to specific targets, aptamer pools that facilitate detection of a biomarker/microvesicle population, reagents such as primers for nucleic acid sequencing or amplification, arrays for nucleic acid hybridization, detectable labels, solvents or buffers and the like, various linkers, various assay components, blockers, and the like (Paragraph 622, lines 1-10). Also, O’Neill teaches that aptamers were then pooled after ligating barcodes and adapter sequences (Paragraph 644, lines 1-2). O’Neill teaches that the previously described method can be applied to create a sequencing library via the Illumina TruSeq Stranded mRNA HT Sample Prep and subjected to HiSeq Rapid paired-end 100 bp plus sequencing on an Illumina HiSeq 2500 Ultra-High- Throughput Sequencing System (Paragraph 1280, lines 5-10).
Regarding claim 6, O’Neill teaches that the previously described isolation or size selection method can incorporate modifications comprising a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., polyalkylene glycol, such as polyethylene glycol) (Figures 3-4; Paragraph 56, lines 1-10). Specifically, O’Neill teaches that the PEG has an average molecular weight ranging from about 20 kD to 80 kD (Pargraph 560, lines 1-4).
Regarding claims 7-9, O’Neill teaches that the previously described isolation or size selection method includes application of the solid particles comprising a salt formed between the amino acid ester or amino acid amide and the protonated oligonucleotide wherein the acidic phosphate groups of the oligonucleotide protonates the amino group of the amino acid ester or amino acid amide, or comprises a salt formed between the oligonucleotide; divalent metal cation; and optional carboxylate, phospholipid, phosphatidyl choline, or sphingomyelin (Paragraph 608, lines 5-10).
Further, O’Neill teaches a binding agent (702), here an aptamer for purposes of illustration, tethered to a substrate (701) where the binding agent (702) can be covalently attached to substrate (701) or alternatively the binding agent (702) may also be non-covalently attached (i.e., binding agent (702) can comprise a label which can be attracted to the substrate, such as a biotin group which can form a complex with an avidin/streptavidin molecule that is covalently attached to the substrate (Figure 7; Paragraph 60, lines 1-5). Specifically, O’Neill teaches that the pH and/or ionic strength of the solution can be adjusted with various acids, bases, buffers or salts, including without limitation sodium chloride (NaCl), phosphate-buffered saline (PBS), tris-buffered saline (TBS), sodium phosphate, potassium chloride, potassium phosphate, sodium citrate and saline-sodium citrate (SSC) buffer (Paragraph 236, lines 1-10).
Additionally, O’Neill teaches various useful modifications selected from the group consisting: of a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., the non-immunogenic, high molecular weight compound is polyalkylene glycol, such as polyethylene glycol) (Paragraph 256, lines 1-10). Specifically, O’Neill teaches that the PEG has an average molecular weight ranging from about 20 kD to 80 kD (Paragraph 560, lines 1-4).
Further, O’Neill teaches that the previously described array can incorporate capture agents that can also be attached to beads that can be manipulated to move through the microfluidic channels and are attached to magnetic beads (Paragraph 184, lines 5-10).
Regarding claim 10, O’Neill teaches that the previously described isolation or size selection method includes kits comprising one or more reagent to carry out the methods where the one or more reagent comprises a library of potential binding agents that comprises one or more of an aptamer, antibody, and other useful binding agents described herein or known in the art (Paragraph 341, lines 1-2). Additionally, O’Neill teaches various useful modifications selected from the group consisting: of a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., the non-immunogenic, high molecular weight compound is polyalkylene glycol, such as polyethylene glycol) (Paragraph 256, lines 1-10). Specifically, O’Neill teaches that the PEG has an average molecular weight ranging from about 20 kD to 80 kD (Paragraph 560, lines 1-4). Specifically, O’Neill teaches that the PEG may be used at any appropriate concentration, including where the PEG can be used at a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% to isolate the microvesicles (i.e., the PEG is used at a concentration of 6%) (Paragraph 357, lines 1-5).
Regarding claim 11, O’Neill teaches that these previously described capture arrays can be customized into vesicles that can be isolated from a biological sample using a system that uses multiple antibodies that are specific to the most abundant proteins found in a biological sample, such as blood and that such a system can remove up to several proteins at once, thus unveiling the lower abundance species such as cell-of-origin specific vesicles (Paragraph 225, lines 1-5).
Regarding claim 13, O’Neill teaches that the previously described isolation or size selection method can be applied via the vesicle membrane can be disrupted using mechanical forces, chemical agents, or a combination thereof, where disrupting the microvesicle comprises use of one or more of a detergent, a surfactant, a solvent, an enzyme, or any useful combination thereof (Paragraph 367, lines 1-3). Additionally, O’Neill teaches that the sequences provided herein can also be modified as desired so long as the functional aspects are still maintained (e.g., binding to various targets or ability to characterize a phenotype), where the oligonucleotides may comprise DNA or RNA, incorporate various non-natural nucleotides, incorporate other chemical modifications, or comprise various deletions or insertions and such modifications may facilitate synthesis, stability, delivery, labeling, etc, or may have little to no effect in practice )(Paragraph 440, lines 5-10). Further, O’Neill teaches that in some cases, some nucleotides in an oligonucleotide may be substituted while maintaining functional aspects of the oligonucleotide and similarly, 5’ and 3’ flanking regions may be substituted or still in other cases, only a portion or fragment of an oligonucleotide may be determined to direct its functionality such that other portions can be deleted or substituted (Paragraph 440, lines 10-15).
Regarding claims 14-19, O’Neill teaches that the isolation or size selection methodology can incorporate modifications comprising a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., polyalkylene glycol, such as polyethylene glycol) (Figures 3-4; Paragraph 56, lines 1-10). Further, O’Neill teaches a binding agent (702), here an aptamer for purposes of illustration, tethered to a substrate (701) where the binding agent (702) can be covalently attached to substrate (701) or alternatively the binding agent (702) may also be non-covalently attached (i.e., binding agent (702) can comprise a label which can be attracted to the substrate, such as a biotin group which can form a complex with an avidin/streptavidin molecule that is covalently attached to the substrate (Figure 7; Paragraph 60, lines 1-5). Specifically, O’Neill teaches that the pH and/or ionic strength of the solution can be adjusted with various acids, bases, buffers or salts, including without limitation sodium chloride (NaCl), phosphate-buffered saline (PBS), tris-buffered saline (TBS), sodium phosphate, potassium chloride, potassium phosphate, sodium citrate and saline-sodium citrate (SSC) buffer (Paragraph 236, lines 1-10). Specifically, O’Neill teaches that in order to examine targeted nucleic acids, indicated salt concentrations can be applied for optimal results (i.e., 1 M NaCl) (Figure 18D; Paragraph 1175, lines 1-5).
O’Neill teaches that the previously described isolation or size selection method includes removal of highly abundant proteins and other non-desired entities that can further be facilitated with a non-stringent size exclusion step, where the sample can be processed using a high molecular weight cutoff size exclusion step to preferentially enrich high molecular weight vesicles apart from lower molecular weight proteins and other entities (Paragraph 223, lines 1-5). Further, O’Neill teaches that if a vesicle population is detected or isolated using two, three, four or more binding agents in a differential detection or isolation of a vesicle from a heterogeneous population of vesicles, the particular binding agent profile for the vesicle population provides a biosignature for the particular vesicle population (Paragraph 253, lines 15-20).
Further, O’Neill teaches that the previously described array can incorporate capture agents that can also be attached to beads that can be manipulated to move through the microfluidic channels and are attached to magnetic beads (Paragraph 184, lines 5-10).
O’Neill also teaches that one or more reagents can be the one or more aptamer, a buffer, blocker, enzyme, or combination thereof, and the one or more reagents may comprise any useful reagents for carrying out the subject methods, including without limitation aptamer libraries, substrates such as microbeads or planar arrays or wells, reagents for biomarker and/or microvesicle isolation (i.e., via chromatography, filtration, ultrafiltration, centrifugation, ultracentrifugation, flow cytometry, affinity capture (i.e., to a planar surface, column or bead), polymer precipitation, and/or using microfluidics), aptamers directed to specific targets, aptamer pools that facilitate detection of a biomarker/microvesicle population, reagents such as primers for nucleic acid sequencing or amplification, arrays for nucleic acid hybridization, detectable labels, solvents or buffers and the like, various linkers, various assay components, blockers, and the like (Paragraph 622, lines 1-10). Specifically, O’Neill teaches that the PEG has an average molecular weight ranging from about 20 kD to 80 kD (Paragraph 560, lines 1-4).
Also, O’Neill teaches that aptamers were then pooled after ligating barcodes and adapter sequences (Paragraph 644, lines 1-2). O’Neill teaches that the previously described method can be applied to create a sequencing library via the Illumina TruSeq Stranded mRNA HT Sample Prep and subjected to HiSeq Rapid paired-end 100 bp plus sequencing on an Illumina HiSeq 2500 Ultra-High- Throughput Sequencing System (Paragraph 1280, lines 5-10).
Additionally, O’Neill teaches that the targeted or bound oligos or antibodies can be eluted, captured via their biotin linkage and then combined again with normal biological sample where the unbound oligos are then added again to disease-derived biological sample and isolated (Figure 4, Paragraph 57, lines 1-5). O’Neill also teaches that these capture arrays can be customized into vesicles that can be isolated from a biological sample using a system that uses multiple antibodies that are specific to the most abundant proteins found in a biological sample, such as blood and that such a system can remove up to several proteins at once, thus unveiling the lower abundance species such as cell-of-origin specific vesicles (Paragraph 225, lines 1-5).
Regarding claim 20, O’Neill teaches that the previously described isolation or size selection method can incorporate modifications comprising a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., polyalkylene glycol, such as polyethylene glycol) (Figures 3-4; Paragraph 56, lines 1-10). Specifically, O’Neill teaches that the PEG has an average molecular weight ranging from about 20 kD to 80 kD (Paragraph 560, lines 1-4).
Regarding claims 25 and 26, O’Neill teaches a composition comprising such isolated oligonucleotide or plurality of oligonucleotides (Paragraph 23, lines 1-2). Further, O’Neill teaches that the isolation or size selection methodology can incorporate modifications comprising a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position of the nucleic acid; and any combination thereof, where the modification can be selected from the group consisting of: incorporation of a modified nucleotide, 3’ capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, labeling with a radioisotope, and any combination thereof (i.e., polyalkylene glycol, such as polyethylene glycol) (Figures 3-4; Paragraph 56, lines 1-10). Further, O’Neill teaches a binding agent (702), here an aptamer for purposes of illustration, tethered to a substrate (701) where the binding agent (702) can be covalently attached to substrate (701) or alternatively the binding agent (702) may also be non-covalently attached (i.e., binding agent (702) can comprise a label which can be attracted to the substrate, such as a biotin group which can form a complex with an avidin/streptavidin molecule that is covalently attached to the substrate (Figure 7; Paragraph 60, lines 1-5). Specifically, O’Neill teaches that the pH and/or ionic strength of the solution can be adjusted with various acids, bases, buffers or salts, including without limitation sodium chloride (NaCl), phosphate-buffered saline (PBS), tris-buffered saline (TBS), sodium phosphate, potassium chloride, potassium phosphate, sodium citrate and saline-sodium citrate (SSC) buffer (Paragraph 236, lines 1-10).
O’Neill does not teach or suggest the specific PEG/salt-size-selection conditions used to separate larger adapter-ligated nucleic acid molecules from smaller adapter monomers, nor does O’Neill teach or suggest carboxylated magnetic particles as recited in the amended claims.
Sauer teaches that functional genomics is facilitated by the ability to express genes in heterologous systems and in some cases, function can be assayed by generation of in vitro transcripts of the unknown genes and expressing those transcripts in various expression systems. Plasmids bearing phage promoters are used to generate in vitro transcript and therefore, it is important to ensure that the template plasmid DNA is not contaminated with RNase from the isolation procedure (Abstract). Specifically, they have developed a plasmid purification protocol that does not utilize RNase yet yields pure plasmid DNA where the protocol combines the selective precipitation of RNA with 1.4 M CaCl2, followed by a final selective precipitation of the plasmid DNA in a 10% polyethylene glycol (PEG), 250 mM NaCl solution (Abstract). Further, Sauer teaches that size-selective precipitation of DNA fragments with polyethylene glycol (PEG) is a long-established but underappreciated way of purifying DNA and by decreasing the final PEG concentration in the presence of MgCl2, Hartley and Bowen demonstrated that they could selectively precipitate the larger fragments of a 50-bp DNA molecular weight marker (Introduction: Paragraphs 1-2). Further, Sauer teaches that they tested the use of salt in combination with PEG to precipitate the plasmid DNA (Fig. 2) and observed that in the absence of salt, 10% PEG did not precipitate the plasmid DNA (lane 2), all the nucleic acids are left in the supernatant (lane 3) (Plasmid DNA precipitation with polyethylene glycol).
Shan teaches that given the widespread use of plasmid DNA (pDNA) in biology and medicine, the development of rapid, simple, and robust pDNA extraction methods is increasingly important, where magnetic separation techniques, which employ magnetic particles as both a solid phase adsorbent and a magnetic carrier for pDNA, offer several advantages in terms of processing time and economic cost and besides, these methods also reduce chemical inputs while improving ease of manipulation and automation relative to conventional methods requiring centrifugation, precipitation, filtration, and chromatographic separations (Introduction: Paragraphs 1-2). Specifically, Shan teaches that, the relative amount of supercoiled pDNA extracted by the described magnetic method exceeds that of the standard isopropanol precipitation method, indicating that pDNA conformational changes can be minimized by replacing centrifugation with magnetic separation and similar results were found when RNase-treated crude lysates were used for adsorption experiments (Fig. 1B); Introduction: Paragraphs 6-8).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the nucleic acid isolation, adapter ligation, and sequencing library preparation methods of O’Neill with the PEG-mediated size-selective precipitation methods taught by Sauer. O’Neill recognizes the desirability of isolating and enriching desired nucleic acid populations, including aptamer and nucleic acid species for sequencing applications, and further teaches the use of PEG, magnetic beads, adapter ligation, and sequencing workflows. Sauer teaches that PEF in combination with salt provides selective precipitation and size-based separation of nucleic acid molecules, including preferential recovery of larger DNA fragments while smaller nucleic acid species remain in solution. One of ordinary skill in the art would have been motivated to incorporate Sauer’s PEG size selection methodology into O’Neill’s nucleic acid processing workflow in order to improve removal of undesired smaller nucleic acid species and enrich desired nucleic acid molecules prior to downstream sequencing and analyses, yielding the predictable result of enhanced nucleic acid purification and size selection. Such modification would merely involve the application of a known size-selection technique to a known nucleic acid preparation process and would have had a reasonable expectation of success because both references are directed to nucleic acid purification, enrichment and recovery using PEG-based methodologies.
Further, it would have been obvious to employ the carboxyl-functionalized magnetic particles taught by Shan in the modified O’Neill/Sauer process because Shan teaches that such particles provide an effective solid phase for nucleic acid adsorption, purification, and magnetic separation. Substituting O’Neill’s generic magnetic beads or magnetic capture substrates with the known carboxylated magnetic particles of Shan would have represented the use of a known equivalent magnetic separation substrate to obtain predictable nucleic acid capture and recovery results.
Moreover, to the extend the claimed concentrations of molecular weight ranges differ from the specific examples disclosed in the prior art, optimization of such result-effective variables would have been obvious to one of ordinary skill in the art. Sauer teaches that nucleic acid size-selection is governed by PEG molecular weight and PEG/salt concentrations, and therefore recognizes these parameters as affecting the efficiency and selectivity of nucleic acid recovery. Routine optimization of known result-effective variables to obtain desired size-selection and purification performance constitutes no more than ordinary skill in the art. See MPEP 2144.05; see also in re Aller, 220 F. 2d 454, 456 (CCPA 1955).
Conclusions
No claim is 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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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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