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
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 4/6/2026 has been entered.
Claim Status
Claims 36-37, 42, 49, 49, 50 and 55 were amended (4/6/2026). No new matter was added. Claims 36-55 are under examination (4/6/2026).
Priority
Claims 36-55 receive a priority date of 11/15/2019, the effective filing date of Japanese Provisional Patent JP2019-207329. All priority documents have been received.
Objections Withdrawn
The objection to claims 37-38 due to improper dependence is withdrawn in view of Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 112 (b)
The rejections to claims 36, 39-55 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), are withdrawn due to Applicant’s amendments of claims 36, 42 and 49.
Claim Rejections - 35 USC § 102
The rejection to claims 36-55 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Domenyuk et al., (WO 2017/161357 A1, published 9/21/2017) is withdrawn due to Applicant’s amendments of independent claims 36, 42 and 49. Specifically, Domenyuk does not teach the amended requirement of a library comprising extracellular vesicles that themselves contain a fusion protein and barcode RNA bound to the fusion protein (i.e., the now claimed internal fusion-protein/barcode-RNA arrangement within the extracellular vesicles).
New Rejections
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 36-55 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The claims are drawn to a judicial exception because they recite steps of detecting barcode RNAs, comparing quantity ratios of detected barcode RNAs with corresponding quantity ratios in extracellular vesicles, identifying a barcode RNA having a change in quantity ratio, and identifying a factor from sequence information comprised in the identified barcode RNA. These limitations recite an evaluation and comparison of information, followed by identifying a result based on that information, which is an abstract idea, namely a mental process that could practically be performed in the human mind or with pen and paper, and falls within mathematical concepts to the extent the claims require comparing quantity ratios.
The integration of the judicial exception into the claims does not render them patent eligible because the claims are written at a high level of generality and merely use well-known, routine, and conventional techniques in the field.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES.
The claims provide for a method comprising:
providing a library comprising extracellular vesicles, the extracellular vesicles comprising a fusion protein and a barcode RNA bound to the fusion protein, wherein the step of providing the library comprises:
introducing into extracellular vesicle-secreting cells a nucleic acid comprising a sequence encoding an extracellular vesicle-existing protein and a sequence encoding an RNA-binding domain, and expressing the fusion protein encoded thereby;
introducing multiple expression vectors expressing barcode RNAs, or multiple barcode RNAs, into the extracellular vesicle-secreting cells expressing the fusion protein
producing the extracellular vesicles comprising the fusion protein by culturing the extracellular vesicle-secreting cells in culture fluid; and
collecting the extracellular vesicles from the culture fluid to provide the library comprising extracellular vesicles, wherein the barcode RNA binds to the RNA-binding domain in the fusion protein and is thereby contained in the extracellular vesicles;
administering the library comprising extracellular vesicles to a subject, cell, tissue, or body fluid;
isolating tissue, body fluid, or cells and/or extracting RNAs;
detecting barcode RNAs from the extracted RNAs; and
comparing a quantity ratio of each barcode RNA detected with a quantity ratio of the corresponding barcode RNA in the extracellular vesicles provided in step (a) to identify a barcode RNA having a change in quantity ratio, and identifying a factor from sequence information comprised in the identified barcode RNA.
Thus, the claims are directed to statutory categories (i.e., processes).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES.
Abstract ideas have been identified by the courts by way of example, including fundamental economic practices, certain methods of organizing human activities, an idea ‘of itself,’ and mathematical relationships/formulas. The claims recite a judicial exception. The “mental process” of determining if values exceed thresholds and making decisions based on statistical measures corresponds “an abstraction” (an idea having no particular concrete or tangible form). The mathematical concepts involving statistical measures and threshold comparisons are abstract ideas. Thus, the claimed invention describes a judicial exception, which correspond to abstractions (ideas, having no particular concrete or tangible form) and mathematical relationships.
Step 2A, Prong Two — Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO.
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. However, absent are any additional elements recited in the claim beyond the judicial exceptions which integrate the exception into a practical application of the exception. The “integration into a practical application” requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception.
The claim limitations are considered to be; (a) a mental process of evaluating information by comparing a quantity ratio of each barcode RNA detected with a quantity ratio of the corresponding barcode RNA in the extracellular vesicles to identify a change in quantity ratio, and (b) the additional evaluative step of identifying a factor from sequence information comprised in the identified barcode RNA. These steps amount to observation, evaluation, and analysis of information (i.e., abstract ideas).
While the claims recite steps of providing a library comprising extracellular vesicles, including introducing nucleic acids into extracellular vesicle-secreting cells, expressing a fusion protein, introducing barcode RNAs, culturing the cells, collective extracellular vesicles, administering the library to a subject, cell, tissue or body fluid, extracting RNAs, and detecting barcode RNAs, these additional steps do not integrate the judicial exception into a practical application. Rather, these steps are recited at a high level of generality and amount to mere data gathering and pre-solution activity used to obtain information for the comparison and identification steps.
There are no additional steps which apply either of the identified judicial exceptions into a practical application. Thus, the claims do not provide for any element/step that integrates the law of nature into a practical application. Specifically, the claims do not recite any particular improvement in extracellular vesicle technology, RNA-loading technology, RNA detection technology, or any other laboratory technology. For example, the claims broadly recite an extracellular vesicle-existing protein, an RNA-binding protein comprising an RNA-binding domain, multiple expression vectors expressing barcode RNAs, or multiple barcode RNAs, and detecting barcode RNAs from extracted RNAs, but do not require and particular technical manner of detection or any specific improvement in how extracellular vesicles are produced, loaded, administered or analyzed. Instead, the additional elements merely use conventional laboratory activity as tools to collect information that is then analyzed through the abstract comparison and identification steps.
As a result, there are no additional steps that apply the identified judicial exception in a manner that imposes a meaningful limit on the exception. Thus, the claims do not integrate the abstract idea into a practical application.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO.
The Supreme Court has identified a number of considerations for determining whether a claim with additional elements amounts to “significantly more” than the judicial exception(s) itself. The claims as a whole are analyzed to determine whether any additional element/step, or combination of additional elements/steps, in addition to the identified judicial exception(s) is sufficient to ensure that the claim amounts to “significantly more” than the exception(s).
However, the additional elements of the instant application, individually and in combination, do not amount to “significantly more.” Under the Step 2B analysis, the “physical” elements/steps of, introducing into extracellular vesicle-secreting cells a nucleic acid, expressing the fusion protein, introducing multiple expression vectors expressing barcode RNAs or multiple barcode RNAs, culturing the extracellular vesicle-secreting cells in culture fluid, collecting the extracellular vesicles from the culture fluid, administering the library, extracting RNAs, and detecting barcode RNAs. However, these are merely physical steps for implementing the abstract idea and are recited at ahigh level of generality.
For example, Domenyuk et al., (WO 2017/161357 A1, published 9/21/2017), teaches methods and compositions to identify oligonucleotides that bind targets of interest, where the targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases and the oligonucleotides can be used in diagnostic and therapeutic applications (Abstract). Specifically, Domenyuk teaches that the previously described methodology when applied to nucleic acid ligands containing modified nucleotides, incorporates improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics (Paragraph 208, lines 10-15). Further, Domenyuk teaches that via the library, the at least one oligonucleotide or the plurality of oligonucleotides can be administered to a subject prior to the detecting, where such a method may allow imaging of at least one cell or tissue in the subject (Paragraph 501, lines 1-5), thus establishing these forms of library and administration techniques as conventional.
Further, Sutaria et al. (“Low active loading of cargo into engineered extracellular vesicles results in inefficient miRNA mimic delivery”, Journal of Extracellular Vesicles, 2016) discloses, that extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition and their goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs, where specifically HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein and the gene encoding pre-miR-199a was inserted into an artificial intron of the Lamp2a fusion protein (Abstract). Further, Sutaria teaches that two methods exist to deliver cargo RNAs to EVs and include exogenous (also known as chemical) loading, in which the oligo is introduced into the EVs and endogenous (i.e. active) loading where the EV producing cells synthesizes both the delivery system and the RNA cargo, and specifically developed an active delivery modality that exploits the HIV-1 TAR RNA-TAT peptide interaction by swapping the wild type pre-miR-199a loop with the TAR RNA loop (Introduction: Paragraphs 4-5). Further, Sutaria teaches that the modified pre-miR-199a is designed to recognize the TAT peptide that was introduced into the EVs using a Lamp2a fusion protein and the loading of the miR-199a into EVs was enhanced using this TAT–TAR interaction (Introduction: Paragraphs 4-5). These detailed and specified EV-centered methodologies demonstrate that practitioners were well-versed in analyzing complex interaction data from a plethora of EV-based systems.
Further, Iniguez et al. (“Human MMM induces the release of inactive cofactor and restores methyl malonyl-CoA mutase activity through their complex formation”, Biochimie, published 2017, from IDS 5/12/2022) discloses that human mitochondrial methylmalonic-CoA mutase is an isomerase that converts methyl malonyl-CoA, a crucial step for the incorporation of some compounds derived from the diet into the central metabolism and previously it was demonstrated that hMCM loses activity during catalysis and that the interaction with human MMAA, a GTPase protein, avoided the loss or restored hMCM activity (Abstract). Further, Iniguez teaches that it has been reported that other dependent isomerases such as bacterial glycerol- and diol-dehydratases undergo rapid suicidal inactivation during catalysis and to recover their activities, it was necessary for reactivases to intervene and to demonstrate release the damaged cofactor as a reactivase, an inactive version was generated using OH2Cbl, which is the final form of the cofactor that is generated as a result of oxidative inactivation (Figure 2; Section 3.3), thus establishing a high level of routine and convention for the use of statistical comparisons across various domains.
Therefore, introducing into extracellular vesicle-secreting cells a nucleic acid, expressing the fusion protein encoded thereby, introducing multiple expression vectors expressing barcode RNAs or multiple barcode RNAs, culturing the extracellular vesicle-secreting cells in culture fluid, collecting the extracellular vesicles from the culture fluid, administering the library comprising extracellular vesicles, extracting RNAs, and detecting barcode RNAs were routine and conventional before the effective filing date of the claimed invention.
Simply appending routine and conventional activities previously known to the industry specified at a high level of generality to the judicial exception and/or generally linking the use of the judicial exception(s) to a particular technological environment or field of use, are not found to be enough to qualify as “significantly more.” Nothing is added by identifying the techniques to be used (i.e., providing a library comprising extracellular vesicles, introducing into extracellular vesicle-secreting cells a nucleic acid comprising a sequence encoding an extracellular vesicle-existing protein and a sequence encoding an RNA-binding protein comprising an RNA-binding domain, introducing multiple expression vectors expressing barcode RNAs or multiple barcode RNAs, collecting the extracellular vesicles from the culture fluid, administering the library, extracting RNAs, and detecting barcode RNAs) because those techniques were well-understood, routine, and conventional techniques that a practitioner would have thought of when instructed to prepare extracellular vesicles, load RNA cargo, administer the vesicles, recover RNAs, and analyze the resulting information.
In context with the other recited claim limitations, the language “comparing a quantity ratio of each barcode RNA detected in step (4) with a quantity ratio of the corresponding barcode RNA in the extracellular vesicles provided in step (1) to identify a barcode RNA having a change in quantity ration, and identifying the factor that influences [or affects] efficiency of targeting, kinetics and/or stability, or secretion, from sequence information comprised in the identified barcode RNA” indicates whether or not the relationship/correlation exists between detected barcode RNA ratios and the identified factor.
This information simply tells a practitioner about the relevant informational relationship between the measured barcode RNA ratios and the sequence information associated with a factor, and does not recite any technological improvement in the production, loading, administration, recovery, or detection of extracellular vesicles or RNAs. Thus, when viewed both individually and as an ordered combination, the claimed elements/steps in addition to the identified judicial exception are found insufficient to supply an inventive concept because the elements/steps are considered conventional and specified at a high level of generality. The claim limitations do not transform the abstract idea that they recite into patent-eligible subject matter because “the claims simply instruct the practitioner to implement the abstract idea with routine, conventional activity.”
Accordingly, the claims do not qualify as patent-eligible subject matter.
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 36-55 are rejected under 35 U.S.C. 103 as being unpatentable over Domenyuk et al., (WO 2017/161357 A1, published 9/21/2017) and Sutaria et al. (“Low active loading of cargo into engineered extracellular vesicles results in inefficient miRNA mimic delivery”, Journal of Extracellular Vesicles, 2016).
Regarding claim 36, Domenyuk teaches methods and compositions to identify oligonucleotides that bind targets of interest, where the targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases and the oligonucleotides can be used in diagnostic and therapeutic applications (Abstract). Specifically, Domenyuk teaches that the previously described methodology when applied to nucleic acid ligands containing modified nucleotides, incorporates improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics (Paragraph 208, lines 10-15).
Further, Domenyuk teaches that the previously described methodology provides a method for identifying binding agents comprising contacting a plurality of extracellular microvesicles with a randomly generated library of binding agents, identifying a subset of the library of binding agents that have an affinity to one or more components of the extracellular microvesicles, where the binding agents may comprise aptamers, antibodies, and/or any other useful type of binding agent disclosed herein or known in the art (Paragraph 368, lines 5-15).
Domenyuk also teaches that the previously described screening methodology as applied to extracellular vesicles includes; a method for identifying a plurality of target ligands comprising, (a) contacting a reference microvesicle population with a plurality of ligands that are capable of binding one or more microvesicle surface markers, (b) isolating a plurality of reference ligands, wherein the plurality of reference ligands comprise a subset of the plurality of ligands that do not have an affinity for the reference microvesicle population; (c) contacting or fusing one or more test microvesicle with the plurality of reference ligands; and (d) identifying a subset of ligands from the plurality of reference ligands that form complexes with a surface markers or barcodes on the one or more test microvesicle, thereby identifying the plurality of target ligands (Paragraph 369, lines 1-10). Specifically, Domenyuk teaches that via the library, the at least one oligonucleotide or the plurality of oligonucleotides can be administered to a subject prior to the detecting, where such a method may allow imaging of at least one cell or tissue in the subject (Paragraph 501, lines 1-5).
Regarding claims 37-38, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles can be applied via detecting microvesicles using vesicle surface antigens where a commonly expressed vesicle surface antigen can be CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, Annexm V or MFG- E8; including tetraspanms, a family of membrane proteins with four transmembrane domains (i.e., CD151, CD53, CD37, CD82, CD81, CD9 and CD63) (Paragraph 135, lines 1-5).
Regarding claim 39, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles includes multiple capture binding agents in distinguishable addresses on an array or different wells of an immunoassay plate where the detection binding agents can be against the same antigen as the capture binding agent, or can be directed against other markers (Paragraph 142, lines 10-15). Specifically, Domenyuk teaches that the capture binding agent can be any useful binding agent, i.e., tethered aptamers, antibodies or lectins, and/or the detector antibodies can be similarly substituted, i.e., with detectable or labeled aptamers, antibodies, lectins or other binding proteins or entities (i.e., PMS2) (Table 3; Paragraph 142, lines 10-20).
Regarding claims 40-41, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles can be applied to any useful target or label or barcode, including without limitation a tissue, a cell, an organelle, a protein complex, a lipoprotein, a carbohydrate, a microvesicle, a virus, a membrane fragment, a small molecule, a heavy metal, a toxin, a drug, a nucleic acid, mRNA, microRNA, a protein-nucleic acid complex, and various combinations, fragments and/or complexes of any of these (Paragraph 400, lines 5-10). Further, Domenyuk teaches that the target or label or barcode can be applied via cellular microarrays (also called transfection microarrays), chemical compound microarrays, and carbohydrate arrays (glycoarrays) comprise biochips that provide high-density immobilized arrays of recognition molecules or sequences (i.e., aptamers or antibodies), where biomarker binding is monitored indirectly (i.e., via fluorescence) (Paragraph 150, lines 5-10).
Regarding claim 42, Domenyuk teaches methods and compositions to identify oligonucleotides that bind targets of interest, where the targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases and the oligonucleotides can be used in diagnostic and therapeutic applications (Abstract). Specifically, Domenyuk teaches that the previously described methodology when applied to nucleic acid ligands containing modified nucleotides, incorporates improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics (Paragraph 208, lines 10-15).
Further, Domenyuk teaches that the previously described methodology provides a method for identifying binding agents comprising contacting a plurality of extracellular microvesicles with a randomly generated library of binding agents, identifying a subset of the library of binding agents that have an affinity to one or more components of the extracellular microvesicles, where the binding agents may comprise aptamers, antibodies, and/or any other useful type of binding agent disclosed herein or known in the art (Paragraph 368, lines 5-15).
Domenyuk also teaches that the previously described screening methodology as applied to extracellular vesicles includes; a method for identifying a plurality of target ligands comprising, (a) contacting a reference microvesicle population with a plurality of ligands that are capable of binding one or more microvesicle surface markers, (b) isolating a plurality of reference ligands, wherein the plurality of reference ligands comprise a subset of the plurality of ligands that do not have an affinity for the reference microvesicle population; (c) contacting or fusing one or more test microvesicle with the plurality of reference ligands; and (d) identifying a subset of ligands from the plurality of reference ligands that form complexes with a surface markers or barcodes on the one or more test microvesicle, thereby identifying the plurality of target ligands (Paragraph 369, lines 1-10). Specifically, Domenyuk teaches that via the library, the at least one oligonucleotide or the plurality of oligonucleotides can be administered to a subject prior to the detecting, where such a method may allow imaging of at least one cell or tissue in the subject (Paragraph 501, lines 1-5).
Regarding claims 43-44, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles can be applied via detecting microvesicles using vesicle surface antigens where a commonly expressed vesicle surface antigen can be CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, Annexm V or MFG- E8; including tetraspanms, a family of membrane proteins with four transmembrane domains (i.e., CD151, CD53, CD37, CD82, CD81, CD9 and CD63) (Paragraph 135, lines 1-5).
Regarding claim 45, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles includes multiple capture binding agents in distinguishable addresses on an array or different wells of an immunoassay plate where the detection binding agents can be against the same antigen as the capture binding agent, or can be directed against other markers (Paragraph 142, lines 10-15). Specifically, Domenyuk teaches that the capture binding agent can be any useful binding agent, i.e., tethered aptamers, antibodies or lectins, and/or the detector antibodies can be similarly substituted, i.e., with detectable or labeled aptamers, antibodies, lectins or other binding proteins or entities (i.e., PMS2) (Table 3; Paragraph 142, lines 10-20).
Regarding claims 46-47, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles can be applied to any useful target or label or barcode, including without limitation a tissue, a cell, an organelle, a protein complex, a lipoprotein, a carbohydrate, a microvesicle, a virus, a membrane fragment, a small molecule, a heavy metal, a toxin, a drug, a nucleic acid, mRNA, microRNA, a protein-nucleic acid complex, and various combinations, fragments and/or complexes of any of these (Paragraph 400, lines 5-10). Further, Domenyuk teaches that the target or label or barcode can be applied via cellular microarrays (also called transfection microarrays), chemical compound microarrays, and carbohydrate arrays (glycoarrays) comprise biochips that provide high-density immobilized arrays of recognition molecules or sequences (i.e., aptamers or antibodies), where biomarker binding is monitored indirectly (i.e., via fluorescence) (Paragraph 150, lines 5-10).
Regarding claim 48, Domenyuk teaches that the previously described method of screening can be applied to proteins, RNA or DNA as appropriate, which can be circulating freely or in a complex with other biological molecules, and as desired, markers (Table 4) can also be used to detect tumor tissue or for capture and/or detection of vesicles for characterizing phenotypes (Paragraph 136, lines 1-5).
Regarding claim 49, Domenyuk teaches methods and compositions to identify oligonucleotides that bind targets of interest, where the targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases and the oligonucleotides can be used in diagnostic and therapeutic applications (Abstract). Specifically, Domenyuk teaches that the previously described methodology when applied to nucleic acid ligands containing modified nucleotides, incorporates improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics (Paragraph 208, lines 10-15).
Further, Domenyuk teaches that the previously described methodology provides a method for identifying binding agents comprising contacting a plurality of extracellular microvesicles with a randomly generated library of binding agents, identifying a subset of the library of binding agents that have an affinity to one or more components of the extracellular microvesicles, where the binding agents may comprise aptamers, antibodies, and/or any other useful type of binding agent disclosed herein or known in the art (Paragraph 368, lines 5-15).
Domenyuk also teaches that the previously described screening methodology as applied to extracellular vesicles includes; a method for identifying a plurality of target ligands comprising, (a) contacting a reference microvesicle population with a plurality of ligands that are capable of binding one or more microvesicle surface markers, (b) isolating a plurality of reference ligands, wherein the plurality of reference ligands comprise a subset of the plurality of ligands that do not have an affinity for the reference microvesicle population; (c) contacting or fusing one or more test microvesicle with the plurality of reference ligands; and (d) identifying a subset of ligands from the plurality of reference ligands that form complexes with a surface markers or barcodes on the one or more test microvesicle, thereby identifying the plurality of target ligands (Paragraph 369, lines 1-10). Specifically, Domenyuk teaches that via the library, the at least one oligonucleotide or the plurality of oligonucleotides can be administered to a subject prior to the detecting, where such a method may allow imaging of at least one cell or tissue in the subject (Paragraph 501, lines 1-5).
Regarding claims 50-51, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles can be applied via detecting microvesicles using vesicle surface antigens where a commonly expressed vesicle surface antigen can be CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, Annexm V or MFG- E8; including tetraspanms, a family of membrane proteins with four transmembrane domains (i.e., CD151, CD53, CD37, CD82, CD81, CD9 and CD63) (Paragraph 135, lines 1-5).
Regarding claim 52, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles includes multiple capture binding agents in distinguishable addresses on an array or different wells of an immunoassay plate where the detection binding agents can be against the same antigen as the capture binding agent, or can be directed against other markers (Paragraph 142, lines 10-15). Specifically, Domenyuk teaches that the capture binding agent can be any useful binding agent, i.e., tethered aptamers, antibodies or lectins, and/or the detector antibodies can be similarly substituted, i.e., with detectable or labeled aptamers, antibodies, lectins or other binding proteins or entities (i.e., PMS2) (Table 3; Paragraph 142, lines 10-20).
Regarding claims 53-54, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles can be applied to any useful target or label or barcode, including without limitation a tissue, a cell, an organelle, a protein complex, a lipoprotein, a carbohydrate, a microvesicle, a virus, a membrane fragment, a small molecule, a heavy metal, a toxin, a drug, a nucleic acid, mRNA, microRNA, a protein-nucleic acid complex, and various combinations, fragments and/or complexes of any of these (Paragraph 400, lines 5-10). Further, Domenyuk teaches that the target or label or barcode can be applied via cellular microarrays (also called transfection microarrays), chemical compound microarrays, and carbohydrate arrays (glycoarrays) comprise biochips that provide high-density immobilized arrays of recognition molecules or sequences (i.e., aptamers or antibodies), where biomarker binding is monitored indirectly (i.e., via fluorescence) (Paragraph 150, lines 5-10).
Regarding claim 55, Domenyuk teaches that the previously described method of screening or identifying extracellular microvesicles can be applied to identify oligonucleotides that bind targets of interest, where the targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases and the oligonucleotides can be used in diagnostic and therapeutic applications (Abstract).
Domenyuk does not teach or suggest cell-based production of extracellular vesicles containing internally loaded barcode RNA via binding of the barcode RNA to an RNA-binding domain of a fusion protein comprising an extracellular vesicle-existing protein.
Sutaria teaches that extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition and the goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs (Abstract). Specifically, Sutaria teaches that HEK293T cells were engineered to produce EVs expressing a lysosomal associated
membrane, Lamp2a fusion protein and the gene encoding pre-miR-199a was inserted into an artificial intron of the Lamp2a fusion protein (Abstract). Further, Sutaria teaches that two methods exist to deliver cargo RNAs to EVs and include exogenous (also known as chemical) loading, in which the oligo is introduced into the EVs and endogenous (i.e. active) loading where the EV producing cells synthesizes both the delivery system and the RNA cargo, and specifically developed an active delivery modality that exploits the HIV-1 TAR RNA-TAT peptide interaction by swapping the wild type pre-miR-199a loop with the TAR RNA loop (Introduction: Paragraphs 4-5). Further, Sutaria teaches that the modified pre-miR-199a is designed to recognize the TAT peptide that was introduced into the EVs using a Lamp2a fusion protein and the loading of the miR-199a into EVs was enhanced using this TAT–TAR interaction (Introduction: Paragraphs 4-5).
Therefore, it would have been obvious to modify Domenyuk’s extracellular vesicle screening platform with Sutaria’s known cell-based EV cargo loading system in order to provide identifiable RNA cargo within the vesicles, because Sutaria teaches engineering EV-producing cells to express a fusion protein and to actively load RNA cargo into the vesicles through a specific RNA-protein interaction, which would have predictably improved tracking and evaluation of vesicle targeting and delivery. And, thus, Sutaria teaches the concept of producing extracellular vesicles in cells using a fusion protein and loading RNA cargo into those vesicles through binding between the RNA cargo and a protein component associated with the fusion construct.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to modify Domenyuk’s extracellular vesicle screening platform with Sutaria’s known cell-based RNA-loading strategy in order to provide identifiable RNA cargo within the extracellular vesicles. A person of ordinary skill in the art would have been motivated to do so because loading RNA cargo into the vesicles would have predictably improved the ability to track, compare, and evaluate vesicle distribution, targeting and delivery, while providing a detectable nucleic acid marker within the vesicles for downstream analysis. The combination would have involved the predictable use of a known EV cargo-loading technique, taught by Sutaria, in the known EV screening context of Domenyuk, and would therefore have been obvious. Moreover, one of ordinary skill in the art would have had a reasonable expectation of success in making this combination because Sutaria expressly teaches that RNA cargo can be actively loaded into extracellular vesicles produced by engineered cells using a fusion-protein/RNA interaction, and Domenyuk already teaches using extracellular vesicle-related libraries for screening and detection purposes. Thus, combining Sutaria’s known RNA-loading approach with Domenyuk’s known extracellular vesicle screening platform would have amounted to the predictable use of prior art elements according to their established functions.
Applicant’s Response: The Applicant argues that the amended independent claims, 36, 42, and 49, are not anticipated by Domenyuk because Domenyuk allegedly does not teach a library of extracellular vesicles themselves, but rather teaches a library of binding agents/oligonucleotides that contact extracellular vesicle surfaces for capture, detection or characterization. The Applicant further argues the newly amended claimed method instead requires extracellular vesicles containing internal barcode RNA bound to a fusion protein, and identifies factors influencing EV properties by changes in barcode RNA quantity, not by external surface binding or capture as in Domenyuk.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are found to be partially persuasive, as discussed below.
As stated above, the Applicant’s arguments are partially persuasive. Specifically, the Applicant is persuasive that Domenyuk alone does not disclose a library of extracellular vesicles themselves comprising a fusion protein and barcode RNA bound to the fusion protein and contained within the vesicles, and for that reason the prior 102 rejection is withdrawn. However, the arguments are not persuasive as to patentability under 103, because nonobviousness cannot be established by attacking Domenyuk individually where the rejection relies on a combination of references. See MPEP 2145 (a).
Domenyuk still teaches the broad screening framework of using extracellular vesicle/microvesicle-related libraries to evaluate targeting, delivery, and detection characteristics, including administration/contacting and downstream identification. Sutaria teaches the missing limitation, namely cell-based production of extracellular vesicles containing internally loaded RNA cargo via fusion protein/RNA interaction, including engineering EV-producing cells to express a Lamp2a fusion protein and using a specific RNA-protein interaction (TAT-TAR) to enhance loading of RNA cargo into EVs. It would have been obvious to modify Domenyuk’s screening platform with Sutaria’s known EV cargo-loading strategy to provide identifiable RNA cargo within the vesicles for tracking and evaluating vesicle targeting and delivery, with a reasonable expectation of success because Sutaria demonstrates that such fusion-protein-mediated RNA loading into EVs works in engineered EV-producing cells. See MPEP 2141, 2143, 2144.
In order to overcome these rejections, Applicant is recommended to further narrow the claims to require a more specific defined localization/configuration of the barcode RNA elative to the fusion protein within the extracellular vesicle, and/or a unique sequence correlation tied to the specific vesicle-producing construct, rather than the current broader language that the RNA is merely “bound” and “contained” in the vesicle.
Conclusion
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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