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 .
Claims Status
Claims 46, 48-51, 53-57, 59-64, 66, & 67 filed on 02/03/2026 are pending. Claim 65 is withdrawn from consideration as being drawn to a non-elected invention. The addition of new claim 67 in the reply filed 02/03/2026 is acknowledged. All the amendments and arguments have been thoroughly reviewed but are deemed insufficient to place this application in condition for allowance. The following rejections are either newly applied, as necessitated by amendment, or are reiterated. They constitute the complete set being presently applied to the instant application. Response to Applicant’s argument follow. This action is FINAL.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Any rejection not reiterated is hereby withdrawn in view of the amendments to the claims.
Claim Rejections - 35 USC § 103
Claim(s) 46, 48, 50, 51, 53-57, 59, 64, 66, & 67 is/are rejected under 35 U.S.C. 103 as being unpatentable over Edelman (Edelman, EP3587589 A1, January 2020), in view of Martin (Martin et al.; ACS Chemical Neuroscience, Vol. 8, pages 356-367, January 39th, 2017).
Regarding newly amended claim 46, Edelman teaches a method of barcoding fixed nucleic acids comprising crosslinking (fixing) biological microparticles, derived from a particular cell or tissue type (paragraph [0012] lines 3-10), prior to appending one or more barcode sequences to one or more target nucleic acid molecules (in a fixed cell or fixed nucleus generating a barcoded nucleic acid molecule using the target nucleic acid molecules and a nucleic acid barcode) (paragraph [0473] lines 1-7; paragraph [0474] lines 1-9; paragraph [0475] lines 1-9). In addition, Edelman teaches that optionally, following a step of crosslinking (fixing), one or more steps of partially or fully reversing the crosslinks may be performed (wherein the fixed cell of the fixed cell nucleus has not been de-crosslinked or un-fixed) (paragraph [0474] lines 7-8; paragraph [0475] lines 6-9).
Edelman fails to teach a method of dissociating the tissue segments after fixation to provide a plurality of cells or cell nuclei, wherein the cells or cell nuclei comprise a plurality of nucleic acid molecules.
Martin teaches a method for dissecting (dividing) frozen brain tissue samples in the presence of a fixing agent (pg. 363 column 2 4th full paragraph lines 2-4; pg. 364 column 1 1st full paragraph lines 1-7; pg. 364 column 1 2nd full paragraph lines 1-6), followed by dissociation of the tissue segments into brain cells followed by recovery and analysis of nucleic acids (pg. 357-358 paragraph bridging pg. 357 & 358 lines 1-15 & 22-23, pg. 359 column 2 2nd full paragraph lines 1-3; pg. 359-360 paragraph bridging pg. 359 & pg. 360 lines 1-2; pg. 360-361 paragraph bridging pg. 360 & 361 lines 1-4 & 7-14; pg.364-365 paragraph bridging pg. 364 & 365 lines 1-12). In addition, Martin teaches that fixing tissue prior to cellular dissociation maintains the integrity of cytoplasmic and extracellular membrane in addition to nucleic acids and these methods can be used with frozen tissue which allows for storage of the tissue until investigators are ready to process it (pg. 360-361 paragraph bridging pg. 360 & 361 lines 7-14).
Edelman and Martin are considered to be analogous to the claimed invention because they are all in the same field of methods for the analysis of cells treated with a fixing agent. Therefore, it would have been prima facie obvious to one of ordinary skill in the art to have modified the method taught in Edelman to incorporate the step of dissociation after fixation taught by Martin for the nucleic acid analysis of a frozen tissue sample because Martin teaches that freezing tissue samples followed by fixation and then dissociation allows for the tissue samples to be stored until ready for processing while still maintaining the integrity of the cells and nucleic acids in the tissue sample.
Regarding claim 48, Edelman teaches the sample may be chemically crosslinked with a chemical crosslinking agent comprising formaldehyde, paraformaldehyde, etc. (fixing agent comprising a cross-linking agent) (paragraph [0285] lines 1-2; paragraph [0286] lines 1-3).
Regarding claim 50, Martin teaches method of dissociating the fixed tissue segments through mincing the tissue segment with two sharp razor blades (mechanical methods) (pg.364-365 paragraph bridging pg. 364 & 365 lines 1-12).
Regarding amended claim 51, Edelman teaches the crosslinking step may be further ended by a quenching step (method further comprises contacting the plurality of fixed cells or fixed nucleic with a quenching medium or quenching solution) (paragraph [0286] lines 1-4).
Regarding claim 53, Martin teaches recovery and analysis of nucleic acids, specifically mRNA, in the fixation and dissociation method for frozen tissue samples (pg. 359-360 paragraph bridging pg. 359 & pg. 360 lines 1-2; pg. 360-361 paragraph bridging pg. 360 & 361 lines 1-4).
Regarding amended claim 54 & claim 55-57, Edelman teaches crosslinking (fixing) biological microparticles, derived from a particular cell or tissue type, prior to appending one or more barcode sequences (plurality of nucleic acid probes) to one or more target nucleic acid molecules (plurality of nucleic acid molecules of the plurality of fixed cells or fixed cell nuclei) (paragraph [0473] lines 1-7; paragraph [0474] lines 1-9; paragraph [0475] lines 1-9) (claim 54). In addition, Edelman teaches that a step of appending one or more barcode sequences (plurality of nucleic acid probes comprising a first probe and a second probe) to one or more target nucleic acid molecules (nucleic acid molecule) comprises annealing of hybridization (first and second probe hybridize to a nucleic acid molecule to form a nucleic acid complex comprising a barcode sequence in the first and second probe) (paragraph [0488] lines 1-3; paragraph [0538] lines 1-2) (claims 55-57).
Regarding amended claim 59, Edelman teaches the use of multimeric barcoding reagents comprises partitioning of the multiple barcode sequences in which the barcode molecules may be linked by a support comprising a plate of wells wherein the barcode molecules are immobilized in different wells of the plate (partitioning plurality of fixed cells or fixed cell nucleic into a plurality of wells) (paragraph [0537] lines 2-11; paragraph [0545] lines 1-3; paragraph [0554] lines 5-8).
Regarding amended claim 64, Edelman teaches the use of multimeric barcoding reagents comprises partitioning of the multiple barcode sequences in which the barcode molecules may be linked by a support comprising a plate of wells wherein the barcode molecules are immobilized in different wells of the plate (plurality of wells comprises wells having no more than one fixed cell or one fixed cell nucleus) (paragraph [0537] lines 2-11; paragraph [0545] lines 1-3; paragraph [0554] lines 5-8).
Regarding claim 66, Edelman teaches the target regions may be used to anneal the barcoded oligonucleotides to the target nucleic acids and then may be used as primers for a primer-extension reaction or a polymerase chain reaction (further comprises performing a nucleic acid extension reaction) (paragraph [0571] lines 1-3).
Regarding new claim 67, Edelman teaches the target regions may be used to anneal the barcoded oligonucleotides to the target nucleic acids and then may be used as primers for a primer-extension reaction or a polymerase chain reaction (comprises generating the barcoded nucleic acid molecule via a polymerase) (paragraph [0571] lines 1-3).
Claim(s) 49 is/are rejected under 35 U.S.C. 103 as being unpatentable over Edelman (Edelman, EP3587589 A1, January 2020) and Martin (Martin et al.; ACS Chemical Neuroscience, Vol. 8, pages 356-367, January 39th, 2017), as applied to claims 46, 48, 50, 51, 53-57, 59, 64, 66, & 67 above, and further in view of Groelz (Groelz et al.; Experimental and Molecular Pathology, Vol. 94, pages 188-194, July 17th, 2012).
The teachings of Edelman and Martin with respect to claim 46 are discussed above.
Regarding claim 49, Edelman and Martin fail to teach the fixing agent comprises formalin and includes about 2% to about 6% by weight formaldehyde in water.
Groelz teaches formalin, a 4% aqueous formaldehyde solution, is the most widely used fixative for preservation of tissue morphology and a method where the tissue samples are fixed with formalin (4% formaldehyde solution) (Pg. 188-189 paragraph bridging pg. 188 & pg. 189 lines 1-3; pg. 189 column 2 1st full paragraph line 1) Groelz also teaches this method of fixation allows for isolation of moderately high numbers of RNA or adequate RNA integrity number (RIN) (pg. 190-191 paragraph bridging pg.190 & pg. 191 lines 1-3).
Edelman, Martin, and Groelz are considered to be analogous to the claimed invention because they are all in the same field of methods for the analysis of cells treated with a fixing agent. Therefore, it would have been prima facie obvious to one of ordinary skill in the art to have modified the fixation methods taught in Edelman and Martin to incorporate use of formalin as a fixing agent as taught by Groelz for the nucleic acid analysis of a frozen tissue sample because Groelz teaches that formalin is a widely used and established method for fixation providing reliability and because Groelz teaches this method of fixation allows for isolation of moderately high numbers of RNA for analysis.
Claim(s) 60-63 is/are rejected under 35 U.S.C. 103 as being unpatentable over Edelman (Edelman, EP3587589 A1, January 2020) and Martin (Martin et al.; ACS Chemical Neuroscience, Vol. 8, pages 356-367, January 39th, 2017), as applied to claims 46, 48, 50, 51, 53-57, 59, 64, 66, & 67 above, and further in view of Belhocine (Belhocine et al.; US Patent Application Publication No. US 2019/0367969).
The teachings of Edelman and Martin with respect to claims 46, 54, & 55 are discussed above.
Regarding claims 60-63, Edelman and Martin fail to teach wherein a cell of the plurality of cells comprises the nucleic acid complex and wherein the cell comprises a labeling agent.
Belhocine teaches an embodiment where the generation of a barcoded nucleic acid molecule comprises linking a first and second probe to yield a probe-linked nucleic acid molecule which can then be barcoded with a barcode sequence of a nucleic acid barcode molecule to provide a barcoded probe-linked nucleic acid molecule (barcoded nucleic acid complex) (paragraph [0457] lines 1-3 & 18-29). Belhocine also teaches the generation of a barcoded nucleic acid molecules (barcoded nucleic acid complex) through use of a reporter oligonucleotide (labelling agent comprises the reporter oligonucleotide) and a nucleic acid barcode molecule (pg. 190 claim 1 lines 6-7 & 18-21). Belhocine also teaches the labelling agent may include an antibody, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, monobody, an affimer, a darpin, and a protein scaffold (paragraph [0416] lines 5-12). Belhocine also teaches that the labelling agent is configured to couple to protein (a feature of the cell) in which the protein is a cell surface protein or is an intracellular protein (Pg. 190 claim 1 lines 7-9; pg. 191 claim 16 lines 1-2; pg. 191 claim 17 lines 1-2). Finally, Belhocine teaches that the labeling agent can provide information on cell surface features as a form of analysis and characterization of a sample (paragraph [0416] lines 1-3).
Edelman, Martin, and Belhocine are considered to be analogous to the claimed invention because they are all in the same field of methods for the nucleic acid analysis of cells. Therefore, it would have been prima facie obvious to one of ordinary skill in the art to prior to the effective filing date, to have modified the method of forming a nucleic acid complex taught in Edelman and Martin to incorporate a labeling agent as taught by Belhocine for the nucleic acid analysis of cell samples because Belhocine teaches that a labelling agent can provide information on the surface features of a cell providing a method to analyze and characterize the cells in a sample.
Response to Arguments
The response traverses the rejection. The response asserts that the asserted combination of Porter and Martin does not meet all of the elements of the claim and the claim 46 has been amended to clarify certain differences between the claimed subject matter that the cited references. Specifically, the response asserts that Porter does not teaches, disclose, or suggest in a fixed cell or fixed cell nucleus that has not been de-crosslinked or un-fixed and generating a barcoded nucleic acid molecule and that Marin does not cure the deficiencies of Porter, as Martin also does not teach, disclose, or suggest this limitation. This argument has been thoroughly reviewed but was not found persuasive. As the teachings of Porter and the combination of Porter and Marin are no longer relied upon, as necessitated by the amendments to claim 46, the teachings of Porter and the combination of Porter and Martin will not be further discussed. Further, the combination of Edelman and Martin, as applied to claim 46 as necessitated by amendment, teach all of the limitations of the currently pending amended claim 46 as discussed above. Specifically, the prior art of Edelman and Martin, as applied to claim 46 as necessitated by amendment, appreciates a method of barcoding fixed nucleic acids comprising crosslinking (fixing) biological microparticles, derived from a particular cell or tissue type (paragraph [0012] lines 3-10), prior to appending one or more barcode sequences to one or more target nucleic acid molecules (paragraph [0473] lines 1-7; paragraph [0474] lines 1-9; paragraph [0475] lines 1-9) in which it is optional to follow a step of crosslinking (fixing) with one or more steps of partially or fully reversing the crosslinks may be performed (wherein the fixed cell of the fixed cell nucleus has not been de-crosslinked or un-fixed) (paragraph [0474] lines 7-8; paragraph [0475] lines 6-9) and not required to decrosslink or unfix the sample.
The response also asserts that Porter teaches away from the limitation of in a fixed cell or fixed cell nucleus that has not been de-crosslinked or un-fixed and generating a barcoded nucleic acid molecule as the context of generating a barcoded nucleic acid in Porter is in the context of a barcoded primer which requires a polymerase to facilitate primer extension using the target nucleic acid as the template and the Porter teaches at paragraph [0213] that crosslinking is incompatible with polymerases. Further, the response asserts that each example of Porter requires a primer and that Porter teaches that crosslinking is incompatible with polymerases and leads to reduced processivity and to solve this problem it is desired that crosslink reversal is completed prior to reverse transcription. Further, the response asserts that the desire to complete crosslink reversal is not only focused on one embodiment of Porter and that Porter teaches away from generation of barcoded nucleic acid molecule without unfixing/de-crosslinking as is claimed. This argument has been thoroughly reviewed but was not found persuasive for the reasons set forth above.
The response also asserts that a person or ordinary skill in the art would have no reasonable expectation of success in arriving at applicant’s invention from Porter and Martin as Porter leads the person of ordinary skill in the art to believe that the crosslinking is incompatible with polymerases and leads to reduced processivity leading to the skilled artisan to have an expectation from Porter that generation of a barcoded nucleic acid molecule without de-crosslinking or un-fixing would be unsuccessful. This argument has been thoroughly reviewed but was not found persuasive for the reasons set forth above.
The response also asserts that claims 48, 50, 53-57, 64, & 66 depend from and include all the elements of claim 46 and recite additional elements of particular advantage and utility and that these claims are not obvious at least for the reasons set for above. This argument has been thoroughly reviewed but was not found persuasive for the reasons set forth above.
The response also asserts that claims 49 and 51 are not obvious over Porter, Martin, and further in view of Greolz at least for the reasons that independent claim 46, from which claims 49 and 51 depend, are not obvious over these references and that Greolz does not cure any of the deficiencies discussed above with respect to Porter and Martin. This argument has been thoroughly reviewed but was not found persuasive for the reasons set forth above.
The response also asserts that claims 60-63 are not obvious over Porter, Martin, and further in view of Belhocine at least for the reasons that independent claim 46, from which claims 60-63 depend, are not obvious over these references and that Belhocine does not cure any of the deficiencies discussed above with respect to Porter and Martin. This argument has been thoroughly reviewed but was not found persuasive for the reasons set forth above.
For these reasons and the reasons already made of record and modified to address the claims as currently amended, the rejections are maintained and applied to the newly amended claims.
Conclusion
Claims 46, 48-51, 53-57, 59-64, 66, & 67 are rejected.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BAILEY C BUCHANAN whose telephone number is (703)756-1315. The examiner can normally be reached Monday-Friday 8:00am-5:00pm ET.
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/BAILEY BUCHANAN/Examiner, Art Unit 1682
/JEHANNE S SITTON/ Primary Examiner, Art Unit 1682
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