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 .
Response to Applicant Reply
This office action is in response to Applicant’s reply of 22 June 2026. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance.
Note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
This action is made FINAL.
Withdrawn Objections and Rejections
The objection to the specification in the office action mailed 27 March 2026 is withdrawn in view of the amendment to the specification filed 22 June 2026.
The rejection of claims 94 and 95 under 35 U.S.C. 112(b) in the office action mailed 27 March 2026 is withdrawn in view of the amendment to the claims filed 22 June 2026. Applicant’s argument that the amended claims have clear antecedent basis for their limitations is persuasive.
The rejection of claims 1, 5, 11, 22, 28, 45, 53, 79-80, and 85 under 35 U.S.C. 102 in the office action mailed 27 March 2026 is withdrawn in view of the amendment to the claims filed 22 June 2026. However, upon further consideration a new ground of rejection of these claims is made under 35 U.S.C. 103. See the Response to Arguments section following the Claim Rejections - 35 USC § 103 section below for an expanded explanation of the withdrawn rejection and new ground of rejection.
Information Disclosure Statement
The information disclosure statement (IDS) filed 22 June 2026 is considered, initialed, and attached hereto.
Claim Status
Claims 1, 3, 5, 11, 15, 22, 28-29, 32-33, 45, 52-53, 79-80, 85, 94-95, and 109-110 are pending and under examination.
Claims 1, 15, 22, 28, 29, 32, 79, 80, 85, 94, and 109 have been amended in the amendment to the claims filed 22 June 2026.
Claims 2, 4, 6-10, 12-14, 16-21, 23-27, 30-31, 34-44, 46-51, 54-78, 81-84, 86-93, and 96-108 are canceled.
Claim Rejections - 35 USC § 103
New - Necessitated by Amendment
Claims 1, 5, 11, 22, 28, 45, 53, 79-80, and 85 are rejected under 35 U.S.C. 103 as being unpatentable over Daugharthy et al. (US 2021/0292834, published 23 September 2021, effectively filed 10 October 2018), herein Daugharthy.
Regarding claim 1, Daugharthy teaches a method for processing a biological sample, comprising: (a) contacting the biological sample with a polymer (“A 3D matrix may be formed in situ throughout the cell and tissue sample, such as through the formation of a hydrogel matrix” [0081]), wherein: the polymer comprises one or more functional groups A (“a reactive group on the 3D matrix” [0085]); the biological sample comprises an amplification product of a probe wherein the amplification product comprises one or more functional groups B (“The nucleic acids (e.g., RNA molecule, cDNA molecule, primer, or probe) described herein may comprise a functional moiety […] A nucleotide analog comprising a functional moiety may be incorporated into a growing chain of the nucleic acid (e.g., cDNA molecule, probe, or primer) during nucleic acid synthesis or an extension reaction” [0085]); and the probe comprises a nucleic acid and is configured to directly or indirectly bind to a target analyte in the biological sample (“Identification by hybridization can comprise hybridizing a detection probe to the sequence to be identified” [0076]); (b) reacting the one or more functional groups A with the one or more functional groups B, thereby coupling the polymer to the amplification product of the probe to form a polymer conjugate (“The nucleic acids can be linked to the 3D matrix by the functional moiety. The functional moiety can be reacted with a reactive group on the 3D matrix through conjugation chemistry” [0085]); removing unreacted polymer from the biological sample (“the synthetic 3D matrix may be partially or substantially cleared of certain species or classes of biomolecules, such as lipids and proteins, as by use of detergent and/or protease reagents. According to some aspects of the present disclosure, the sample can be cleared using a detergent solution, such as Triton-X or SDS. The detergent may interact with the molecules allowing the molecules to be washed out or removed” [0089], per instant specification [0098], washing is a method of removing unreacted polymer); (d) shrinking or expanding the polymer conjugate, thereby respectively shrinking or expanding the amplification product (“the 3D matrix can be an expanding FISSEQ matrix” [0081]). Regarding the recitation in the claim “in a targeted manner”, since the claim and the instant specification do not provide a limiting definition of “a targeted manner”, the broadest reasonable interpretation is that “a targeted manner” encompasses any manner of shrinking/expanding the amplification product wherein the amplification product is targeted for shrinking/expanding, in contrast to approaches that may shrink/expand the amplification product by indiscriminately shrinking/expanding structures that happen to have the amplification product as well as structures that don’t. As the method of Daugharthy specifically targets the amplification product by incorporating functional moieties into the amplification product ([0085]) that are linked to the matrix by reaction of the functional moieties with the reactive groups of the matrix ([0085]), the expansion of the amplification products by the expanding FISSEQ matrix is considered to be in a targeted manner.
However, regarding claim 1, Daugharthy does not explicitly teach that the shrinking or expanding of the polymer conjugate occurs “after removing the unreacted polymer from the biological sample”, as required by the claim. Despite this lack of explicit teaching, it would be obvious to modify the order of Daugharthy to perform the shrinking or expanding of the polymer conjugate after the wash that removes the unreacted polymer, because “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results” (MPEP §2144.04 IV. C.).
Regarding claim 5, Daugharthy teaches the method of claim 1 (see above), wherein the reaction between functional group A and functional group B is a click reaction (“In some embodiments, target molecules and/or index molecules are subjected to amplification reactions” [0107]; “Nucleic acid molecules can be amplified by rolling circle amplification (RCA)” [0108]; “The amplicon may comprise functional linkage groups for tethering to the 3D matrix, such as acrylamide or click-reactive groups” [0109]).
Regarding claim 11, Daugharthy teaches the method of claim 1 (see above), wherein the target analyte is a nucleic acid target analyte, and wherein a primary probe is hybridized to a target sequence in the nucleic acid target analyte (“Methods used to determine the 3D spatial position can comprise […] hybridization” [0076]; “In some embodiments, target molecules and/or index molecules are subjected to amplification reactions” [0107]; “The target molecules may be RNA […] The target molecules may be DNA” [0140]; “The probes may be […] used for nucleic acid amplification described herein” [0152]).
Regarding claim 22, Daugharthy teaches the method of claim 1 (see above), wherein the amplification product comprises a detectable label, or a region that directly or indirectly binds to a detectably labeled probe (“the 3D spatial position can be determined by hybridizing a probe having a detectable label attached thereto to an index sequence” [0076]; “the molecules comprising the index may be generated using amplification” [0107]).
Regarding claim 28, Daugharthy teaches the method of claim 22 (see above), wherein the amplification product comprises a barcode sequence, and wherein the barcode sequence corresponds to the target analyte or a portion thereof in the biological sample (“the 3D spatial position can be determined by hybridizing a probe having a detectable label attached thereto to an index sequence co-localized with the target nucleic acid molecule” [0076] the index sequence is considered a barcode sequence).
Regarding claim 45, Daugharthy teaches the method of claim 1 (see above), wherein shrinking or expanding the polymer conjugate is initiated by contacting the biological sample with a solution, by changing a temperature of the biological sample, and/or by exposing the biological sample to light (“the 3D matrix can be an expanding FISSEQ matrix, such as one comprised substantially of poly(acrylate-co-acrylic acid) (PAA) or Poly(N-isopropylacrylamide) (NIPAM). The matrix comprising NIPAM may be expandable or configured to expand by a change in temperature” [0081]).
Regarding claim 53, Daugharthy teaches the method of claim 1 (see above), wherein the biological sample is a processed or cleared biological sample (“the sample can be cleared” [0089]).
Regarding claim 79, Daugharthy teaches the method of claim 1 (see above), wherein contacting the biological sample with the polymer occurs after generation of the amplification product of the probe in the biological sample (“a method for processing or analyzing a plurality of nucleic acid molecules of a cell or cell derivative, comprising: (a) generating in the cell or cell derivative a synthetic three-dimensional (3D) matrix” [0005]; “the method further comprises, prior to (a), providing a plurality of precursor nucleic acid molecules, and subjecting the plurality of precursor nucleic acid molecules to nucleic acid amplification to generate the plurality of nucleic acid molecules” [0014]; “probes may be used to synthesize or amplify nucleic acids” [0152] therefore, the plurality of nucleic acid molecules generated by nucleic acid amplification are the product of probes and this generation is done before contacting the sample with the polymer).
Regarding claim 80, Daugharthy teaches the method of claim 1 (see above), wherein the amplification product of the probe is generated in situ in the biological sample (“The nucleic acids in the biological sample will be hybridized with a probe […] The hybridized sequence may be ligated or subjected to an extension reaction to circularize the nucleic acid molecule. Following circularization, the nucleic acid molecule can be subjected to RCA to amplify the amount of nucleic acid and increase the signal. In situ sequencing can be performed on the capture nucleic acid molecule” [0172]).
Regarding claim 85, Daugharthy teaches the method of claim 1 (see above), wherein the amplification product of the probe is a rolling circle amplification (RCA) product (RCP) of a circular or circularizable probe or probe set that hybridizes to a DNA or RNA molecule in the biological sample (“The nucleic acids in the biological sample will be hybridized with a probe or a plurality of probes […]. The probe will be constructed such that it can be circularized, for example a molecular inversion probe or padlock probe. […] The hybridized sequence may be ligated or subjected to an extension reaction to circularize the nucleic acid molecule. Following circularization, the nucleic acid molecule can be subjected to RCA to amplify the amount of nucleic acid” [0172]; “A nucleic acid may comprise deoxyribonucleotides and/or ribonucleotides, or analogs thereof” [0034]).
Therefore, in view of Daugharthy, the invention as a whole of claims 1, 5, 11, 22, 28, 45, 53, 79-80, and 85 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Maintained - Modified as Necessitated by Amendment
Claims 3 and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Daugharthy et al. (US 2021/0292834, published 23 September 2021), herein Daugharthy, as applied to claims 1, 5, 11, 22, 28, 45, 53, 79-80, and 85 above.
Regarding claim 3, Daugharthy teaches the method of claim 1 (see 35 U.S.C. 103 rejection above), further comprising initiating polymer-polymer cross-linking to form a polymer network between the polymers (“The 3D matrix may be generated by directing precursors of the 3D matrix in to the biological specimen and subjecting the precursors to crosslinking or polymerization reactions” [0081]). Daugharthy does not explicitly teach that this cross-linking occurs between polymers of the polymer conjugate, which requires that it occurs after the polymer conjugate is formed by coupling the polymer to the probe or product thereof, as recited in claim 1.
However, it would be obvious to modify the order of Daugharthy to contact the biological specimen with a polymer (“directing precursors of the 3D matrix in to the biological specimen” [0081]), then react the functional groups A (“reactive group” [0085]) with the functional groups B (“functional moiety” [0085]) to form a polymer conjugate, then cross-link the polymers of the polymer conjugate to form a polymer network (“subjecting the precursors to crosslinking or polymerization reactions” [0081]), because “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results” (MPEP §2144.04 IV. C.). Therefore, the invention as a whole of claim 3 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Regarding claim 29, Daugharthy teaches the method of claim 1 (see 35 U.S.C. 103 rejection above), further comprising detecting a signal associated with the amplification product or the target analyte by imaging the biological sample using fluorescent microscopy (“target molecules and/or index molecules are subjected to amplification reactions” [0107]; “The target molecules may be RNA […] The target molecules may be DNA” [0140]; “probes may be […] used for nucleic acid amplification” [0152]; “the spatial origin of each index can be determined via fluorescent in situ sequencing (FISSEQ)” [0100]). Though Daugharthy also teaches expansion of the amplification product (see 35 U.S.C. 103 rejection of claim 1), Daugharthy does not explicitly teach an order of steps such that the step of detecting a signal by imaging occurs after shrinking or expansion of the amplification product.
However, it would be obvious to modify the order of Daugharthy to expand the amplification product (“the 3D matrix can be an expanding FISSEQ matrix” [0081]), then detect a signal associated with the amplification product or the target analyte by imaging the biological sample using fluorescent microscopy ([0076] and [0151] in above paragraph), because “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results” (MPEP §2144.04 IV. C.). Therefore, the invention as a whole of claim 29 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Maintained - Modified as Necessitated by Amendment
Claims 15, 32, 94-95, and 109-110 are rejected under 35 U.S.C. 103 as being unpatentable over Daugharthy et al. (US 2021/0292834, published 23 September 2021), herein Daugharthy, as applied to claims 1, 3, 5, 11, 22, 28-29, 45, 53, 79-80, and 85 above, and further in view of Church et al. (in IDS filed 24 January 2024)(US 2019/0194709, published 27 June 2019), herein Church.
Regarding claim 15, Daugharthy teaches the method of claim 11 (see 35 U.S.C. 103 rejection above), wherein the amplification product is a rolling circle amplification (RCA) product (RCP) and nucleotides comprising one or more functional groups B are incorporated into the RCP during RCA (“Nucleic acid molecules can be amplified by rolling circle amplification (RCA)” [0108]; “The amplicon may comprise functional linkage groups for tethering to the 3D matrix […] the functional linkages can be incorporated during amplification using nucleotide analogs” [0109]). Daugharthy also teaches that NIPAM is one possible expandable polymer that can be used in the method (“The matrix comprising NIPAM may be expandable” [0081]). However, Daugharthy does not teach the method wherein the RCP is shrunk. This deficiency is made up for in the teachings of Church.
Regarding claim 15, Church teaches methods of coupling biomolecules to polymers and shrinking or expanding the polymers to respectively shrink or expand the coupled biomolecules (“methods related to processing of a sample so that biomolecules of interest are fixed in a 3-dimensional matrix […] the 3-dimensional matrix can expand or shrink/contract with an external stimulus” [0032]). Church also teaches that the 3-dimensional matrix may be comprised of a NIPAM gel that is able to change volume (i.e. expand or shrink/contract) in response to stimuli (“As used herein, a 3-dimensional matrix may refer to a hydrogel” [0032]; “the hydrogel may be a thermally induced hydrogel. The thermally induced hydrogel may undergo a volume transition in response to thermal stimuli. One example of a thermally induced hydrogel is a NIPAM gel” [0123]).
Regarding claim 32, Daugharthy teaches the method of claim 29 (see 35 U.S.C. 103 rejection above), wherein a signal is detected and wherein the amplification product is expanded. However, Daugharthy does not teach that the expanding happens after the detecting and does not teach shrinking of the amplification product before the detecting. These deficiencies are made up for in the teachings of Church.
Regarding claim 32, Church teaches methods of coupling biomolecules to polymers (as discussed with regard to claim 15) wherein the same polymer can undergo both shrinking and expanding as well as fluorescent imaging (“the hydrogel can be expanded upon an external stimulus […] and a reagent mixture may be flown through after the expansion, and then the hydrogel can be contracted subsequently with an external stimulus” [0129]; “In some embodiments, the method further comprises flowing reagents for fluorescent in situ sequencing (FISSEQ) into the three-dimensional matrix subsequent to said swelling” [0007]). Neither Daugharthy nor Church explicitly teach an order of steps such that the step of detecting occurs after the step of shrinking and the step of expanding occurs after the step of detecting. However, it would be obvious to modify the order of steps of the combination of Daugharthy and Church to shrink the amplification product by shrinking the polymer, then detect the signal, then expand the amplification product by expanding the polymer, because “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results” (MPEP §2144.04 IV. C.).
Regarding claim 94, Daugharthy teaches the method of claim 85 (see 35 U.S.C. 103 rejection above), further comprising degrading the polymer conjugate after detecting a signal associated with the amplification product in the biological sample (“Following circularization, the nucleic acid molecule can be subjected to RCA to amplify the amount of nucleic acid and increase the signal. In situ sequencing can be performed on the capture nucleic acid molecule to identify the SMI sequence. The matrix will be dissolved using enzymatic or chemical reactions to break linkages in the matrix” [0172] sentences correspond to generating the amplification product, detecting a signal, and degrading the polymer conjugate, respectively). However, Daugharthy does not teach that the amplification product is shrunk or that the degrading step happens after the shrinking step. These deficiencies are made up for in the teachings of Church.
Regarding claim 94, Church teaches methods of coupling biomolecules to polymers and shrinking the polymers to shrink the coupled biomolecules (“methods related to processing of a sample so that biomolecules of interest are fixed in a 3-dimensional matrix […] the 3-dimensional matrix can expand or shrink/contract with an external stimulus” [0032]). Neither Daugharthy nor Church explicitly teach an order of steps such that the step of degrading the polymer occurs after the step of shrinking. However, it would be obvious to modify the order of steps of the combination of Daugharthy and Church to shrink the amplification product by shrinking the polymer and to detect a signal, then to degrade the polymer, because “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results” (MPEP §2144.04 IV. C.).
Regarding claim 95, the combination of Daugharthy and Church teach the method of claim 94 (see above). Daugharthy further teaches that the degrading comprises contacting the biological sample with a stripping buffer, changing the temperature of the biological sample, and/or exposing the biological sample to light (“In this example, the proteinase K will be used to dissolve the gel […] For hydrogel embedded matrices, sodium periodate may be used to digest acrylate gels containing DHEBA crosslinker” [0172]; “the nucleic acid material may be linked to the 3D matrix via cleavable linkers and the nucleic acid material can be released from the 3D matrix by cleaving the linkers. The cleavable linkers can be chemically cleavable linkers or photo-cleavable linkers” [0144]).
Regarding claim 109, Daugharthy teaches a method, comprising: (a) generating a rolling circle amplification product (RCP) at a location in a biological sample (“Nucleic acid molecules, such as SMI molecules or endogenous molecules may be amplified using various methods for nucleic acid amplification […] Nucleic acid molecules can be amplified by rolling circle amplification (RCA) […] The resulting population of clonal, or substantially clonal indexing molecules is referred to as the amplicon” [0108]); (b) contacting the biological sample with a polymer (“A 3D matrix may be formed in situ throughout the cell and tissue sample, such as through the formation of a hydrogel matrix” [0081]), wherein: the polymer comprises one or more functional groups A (“a reactive group on the 3D matrix” [0085]), and the RCP comprises one or more functional groups B (“The amplicon may comprise functional linkage groups for tethering to the 3D matrix” [0109]); (c) reacting the one or more functional groups A with the one or more functional groups B, thereby coupling the polymer to the RCP to form a polymer conjugate (“The nucleic acids […] described herein may comprise a functional moiety. The nucleic acids can be linked to the 3D matrix by the functional moiety. The functional moiety can be reacted with a reactive group on the 3D matrix through conjugation chemistry” [0085]); (d) removing unreacted polymer from the biological sample (“the synthetic 3D matrix may be partially or substantially cleared of certain species or classes of biomolecules, such as lipids and proteins, as by use of detergent and/or protease reagents. According to some aspects of the present disclosure, the sample can be cleared using a detergent solution, such as Triton-X or SDS. The detergent may interact with the molecules allowing the molecules to be washed out or removed” [0089], per instant specification [0098], washing is a method of removing unreacted polymer); and (f) detecting a signal associated with the RCP by imaging the biological sample using fluorescent microscopy, thereby detecting the RCP at the location in the biological sample (“target molecules and/or index molecules are subjected to amplification reactions” [0107]; “The target molecules may be RNA […] The target molecules may be DNA” [0140]; “probes may be […] used for nucleic acid amplification” [0152]; “the spatial origin of each index can be determined via fluorescent in situ sequencing (FISSEQ)” [0100]; “FISSEQ library construction […] library construction may involve […] amplifying the original molecule, such as […] rolling circle amplification” [0118]). However, Daugharthy does not teach shrinking the polymer conjugate, thereby shrinking the RCP. This deficiency is made up for in the teachings of Church.
Regarding claim 109, Church teaches methods of coupling biomolecules to polymers and shrinking the polymers to shrink the coupled biomolecules (“methods related to processing of a sample so that biomolecules of interest are fixed in a 3-dimensional matrix […] the 3-dimensional matrix can expand or shrink/contract with an external stimulus” [0032]). Regarding the recitation in the claim “in a targeted manner”, as discussed with regard to claim 1 above, Daugharthy teaches that the amplification product, in this instance an RCP, is targeted by linking it to the matrix via a specific, targeted reaction. Therefore, the shrinking of the RCP by shrinking of the polymer conjugate occurs in a targeted manner.
However, regarding claim 109, neither Daugharthy nor Church explicitly teaches that the shrinking of the polymer conjugate occurs “after removing the unreacted polymer from the biological sample”, as required by the claim. Despite this lack of explicit teaching, it would be obvious to modify the order steps of the combination of Daugharthy and Church to perform the shrinking or expanding of the polymer conjugate after the wash that removes the unreacted polymer, because “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results” (MPEP §2144.04 IV. C.).
Regarding claim 110, the combination of Daugharthy and Church teach the method of claim 109, and both teach expanding the polymer conjugate, thereby expanding the RCP (“the 3D matrix can be an expanding FISSEQ matrix” Daugharthy [0081]; “the 3-dimensional matrix can expand or shrink/contract with an external stimulus” Church [0032]). Neither Daugharthy nor Church explicitly teach an order of steps such that the step of expanding occurs after the step of detecting. However, it would be obvious to modify the order of steps of the combination of Daugharthy and Church to detect the signal, then expand the probe or product thereof by expanding the polymer, because “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results” (MPEP §2144.04 IV. C.).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of shrinking in the method taught by Church for expansion in the method taught by Daugharthy. One of ordinary skill in the art would expect that the results of the substitution would be predictable because Daugharthy and Church both teach methods of analyzing biomolecules by coupling the biomolecules to hydrogels, such as a NIPAM gel, that have their volume changed. Therefore, the invention as a whole of claims 15, 32, 94-95, and 109-110 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Maintained
Claim 33 is rejected under 35 U.S.C. 103 as being unpatentable over Daugharthy et al. (US 2021/0292834, published 23 September 2021), herein Daugharthy, in view of Church et al. (in IDS filed 24 January 2024)(US 2019/0194709, published 27 June 2019), herein Church, as applied to claims 15, 32, 94-95, and 109-110 above, and further in view of Ishidao et al. ("Solvent concentrations of dimethylsulfoxide-water and 1-propanol-water solutions inside and outside poly (N-isopropylacrylamide) gel" Colloid Polym Sci 272, pages 1313–1316 (1994)), herein Ishidao, and Asano et al. ("Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues" Current Protocols in Cell Biology 80, e56 (2018)), herein Asano.
Regarding claim 33, the combination of Daugharthy and Church teach the method of claim 32 (see 35 U.S.C. 103 rejection above) wherein the polymer conjugate expanded comprises poly(N-isopropylacrylamide) (NIPA or NIPAM) (“the 3D matrix can be an expanding FISSEQ matrix, such as one comprised substantially of poly(acrylate-co-acrylic acid) (PAA) or Poly(N-isopropylacrylamide) (NIPAM)” Daugharthy [0081]; “the hydrogel may be a thermally induced hydrogel. The thermally induced hydrogel may undergo a volume transition in response to thermal stimuli. One example of a thermally induced hydrogel is a NIPAM gel” Church [0123]). However, neither Daugharthy nor Church teaches that the polymer conjugate is expanded in a buffer composition comprising at least or about 50% DMSO, a detergent or surfactant, and a salt. These deficiencies are made up for in the combination of the teachings of Ishidao and Asano.
Regarding claim 33, Ishidao teaches that NIPA gels expand in buffer compositions of >90% DMSO (Fig. 1, the filled circles represent DMSO-water solutions where the DMSO concentration ranges from 0% to 100% on the y-axis, when the swelling ratio V/V0 on the x-axis exceeds 100 swelling occurs, at the highest tick mark on the y-axis corresponding to 90% DMSO the swelling ratio is clearly >100). However, neither Daugharthy, Church, nor Ishidao teach a polymer conjugate being expanded in a buffer composition comprising (1) a detergent or surfactant and (2) a salt. This deficiency is made up for in the teachings of Asano.
Regarding claim 33, Asano teaches a method of expanding a gel for expansion microscopy of a nucleic acid target wherein the expansion buffer comprises a detergent or surfactant and a salt (“To expand the samples, wash three times, each time for 10 min with 0.05× SSCT. The expansion factor can be tuned by altering the salt concentration” page 25, step 25; SSCT, 0.05× recipe, page 35, demonstrates that 0.05× SSCT contains Tween-20, a surfactant and detergent, and SSC (saline-sodium citrate), a salt, and that together these are 0.75% volume of a 0.05× SSCT solution).
In view of Asano’s teaching that the expansion factor can be tuned by altering the salt concentration, one of ordinary skill in the art would be motivated to combine the expansion buffer for expanding gels coupled with nucleic acids for expansion microscopy taught by Asano with the method of analyzing samples by coupling nucleic acids with an expandable gel taught by the combination of Daugharthy and Church (the combination of Daugharthy and Church is obvious as discussed in the 35 U.S.C. 103 rejection of claims 15, 32, 94-95, and 109-110 above). This would improve the method of the combination of Daugharthy and Church by allowing the expansion factor to be tuned by the salt concentration, thereby providing motivation to use the buffer of Asano. One of ordinary skill in the art would have a reasonable expectation of success, because Daugharthy, Church, and Asano teach methods of coupling nucleic acids with expandable gels. Therefore, the combination of Daugharthy, Church, and Asano would be obvious to one of ordinary skill in art prior to the effective filing date of the claimed invention.
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of DMSO in the swelling solution of Ishidao for the nuclease-free water in the buffer taught by Asano in the combination of Daugharthy, Church, and Asano. In Figure 1, Ishidao teaches that both components, DMSO and water, are known to have the same function of causing a NIPA gel to swell when the buffer is primarily water (see Figure 1 when the DMSO concentration of the DMSO-water mixture is <10%) and when the buffer is primarily DMSO (see Figure 1 when the DMSO concentration of the DMSO-water mixture is >90%), so both elements and their function were known in the prior art. One of ordinary skill in the art would expect that the results of the substitution of DMSO for water in the buffer would be predictable in that it would maintain the function of the gel being able to expand in the buffer because Daugharthy and Church both teach the gel being a NIPA gel and Ishidao teaches NIPA gels being expanded in both mixtures that are primarily water or primarily DMSO. Furthermore, the salt and detergent taught by Asano only makes up 0.75% of the solution, so one of ordinary skill in the art would be able combine the salt and detergent with the >90% DMSO taught by Ishidao. Therefore, the invention as a whole of claim 33 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Maintained
Claim 52 is rejected under 35 U.S.C. 103 as being unpatentable over Daugharthy et al. (US 2021/0292834, published 23 September 2021), herein Daugharthy, as applied to claims 1, 3, 5, 11, 22, 28-29, 45, 53, 79-80, and 85 above, and further in view of Dewal (in IDS filed 24 January 2024)(WO 2020/142490, published 9 July 2020).
Regarding claim 52, Daugharthy teaches the method of claim 1 (see 35 U.S.C. 103 rejection above), wherein the biological sample is a tissue (“A biological sample may be solid matter (e.g., biological tissue” [0045]). However, Daugharthy does not explicitly teach that the tissue is a tissue section. This deficiency is made up for in the teachings of Dewal.
Regarding claim 52, Dewal teaches a method of expansion microscopy (ExM) wherein a tissue section is coupled with a polymer and expanded by expanding the polymer (“The present invention is an expansion upon a method for optical imaging of biological specimens with resolution better than the classical microscopy diffraction limit, based on physically expanding the specimen itself” [057]; “In this method, cultured cells, fixed tissue, or in principle any other type of sample of interest, including biological materials, are infused with a composition […] that results in it becoming embedded in the sample material, and then the composition can be expanded isotropically” [057]; “In an embodiment of this ExM concept, the composition comprises a polyelectrolyte hydrogel” [058]; “The sample may be a thin section of a frozen or paraffin embedded tissue or other biological specimen section” [063]).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the tissue section taught by Dewal for the tissue in the method taught by Daugharthy. One of ordinary skill in the art would expect that the results of the substitution would be predictable because Daugharthy and Dewal both teach methods of analyzing biomolecules by coupling the biomolecules to expandable hydrogels. Therefore, the invention as a whole of claim 52 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Response to Arguments
Applicant’s arguments, see pages 8-11, filed 22 June 2026, with respect to the rejection of claims 1, 5, 11, 22, 28, 45, 53, 79-80, and 85 under 35 U.S.C. 102 as anticipated by Daugharthy have been fully considered but they are not persuasive.
On pages 8-9 of the reply, Applicant states the ground of rejection under 35 U.S.C. 102, the amendment to step (d) of claim 1, and that this amendment provides a specific mechanism and outcome exemplified in FIG. 5 of the instant application. Applicant further recites relevant teachings of the instant specification and asserts that “the removal of unreacted polymer from the biological sample prior to shrinking or expansion of the polymer conjugate is consistent with, and supports, the targeted shrinking or expansion recited in amended claim 1” (original emphasis). On page 10, Applicant further states the standard for anticipation of a claim. These assertions serve as background for the arguments that follow them in the reply.
On pages 10-11 of the reply, Applicant argues that Daugharthy does not anticipate amended claim 1 because Daugharthy does not disclose “shrinking or expanding a polymer conjugate, thereby respectively shrinking or expanding the amplification product in a targeted manner”. Applicant supports this argument by asserting that claim 1 requires more than expanding a sample-embedded hydrogel or 3D matrix, as taught by Daugharthy, because the claimed dimensional change (shrinking/expanding) is targeted to the amplification product through the polymer conjugate. Applicant further asserts that Daugharthy teaches a dimensional change of the matrix generally, in contrast to the claimed shrinking or expanding of the polymer conjugate itself to shrink or expand the amplification product in a targeted manner.
Daugharthy teaches a 3D matrix (a type of polymer) that is linked to amplification products by specific, targeted reactions, so their expansion by the expandable matrix is considered to occur in a targeted manner. In [0085], Daugharthy teaches that “The nucleic acids can be linked to the 3D matrix by the functional moiety. The functional moiety can be reacted with a reactive group on the 3D matrix through conjugation chemistry […] A nucleotide analog comprising a functional moiety may be incorporated into a growing chain of the nucleic acid (e.g., cDNA molecule, probe, or primer) during nucleic acid synthesis or an extension reaction”. The amplification product is not expanded by the expandable matrix of Daugharthy through general, indiscriminate embedding of nucleic acids into the matrix, but rather is expanded because it is a specific target that is linked to the matrix by the reaction of the incorporated functional groups with reactive groups on the matrix. Therefore, this argument is not persuasive.
On page 11 of the reply, Applicant also argues that Daugharthy does not dislose “removing unreacted polymer from the biological sample” as recited in step (c) of claim 1, because the wash step identified in the rejection, which is recited by Daugharthy in [0089], is directed to clearing a sample of certain biomolecules and is silent as to the removal of unreacted polymer. Applicant further notes, regarding the reference in the office action to [0098] of the instant specification, that the present specification cannot be used to supply a limitation that is absent from a prior art reference per MPEP §2131.
The citation of [0098] of the instant specification is not to supply a limitation, but rather evidence that the removing unreacted polymer from the biological sample is a genus that encompasses the species of washing the biological sample. [0098] of the instant specification states: “In some embodiments, unreacted polymer is removed from the biological sample. In some embodiments, the removing comprises washing the biological sample.” MPEP §2131.02(I) instructs: “"A generic claim cannot be allowed to an applicant if the prior art discloses a species falling within the claimed genus." The species in that case will anticipate the genus. In re Slayter, 276 F.2d 408, 411, 125 USPQ 345, 347 (CCPA 1960)”. Therefore, because washing the biological sample is a species of removing unreacted polymer from the biological sample, the teaching of Daugharthy to wash the sample is the teaching of a species that anticipates the genus limitation of removing unreacted polymer from the biological sample. Furthermore, one of ordinary skill would readily understand that the washing step would remove not just the specified biomolecules, but also other components that are not coupled to the matrix, such as unreacted polymer.
However, Examiner notes that claim 1 (upon which claims 5, 11, 22, 28, 45, 53, 79-80, and 85 depend) as amended in the amendment to the claims filed 22 June 2026 recites that the shrinking or expanding of the polymer conjugate occurs after removing the unreacted polymer from the biological sample, which is an order of steps that is not explicitly taught by Daugharthy, so Daugharthy does not anticipate the amended claim 1 or any claims that depend from claim 1. Therefore, the rejection under 35 U.S.C. 102 has been withdrawn. However, upon further consideration, a new ground of rejection of claims 1, 5, 11, 22, 28, 45, 53, 79-80, and 85 is made under 35 U.S.C. 103 in view of the teachings of Daugharthy. This new ground of rejection of necessitated by the amendment to claim 1 adding the new limitation that step (d) occurs after removing the unreacted polymer from the biological sample.
Applicant’s arguments, see pages 12-13, filed 22 June 2026, with respect to the rejection of claims 3 and 29 under 35 U.S.C. 103 as unpatentable over Daugharthy have been fully considered but they are not persuasive.
On pages 12-13 of the reply, Applicant states the rejections and argues that the Office has not established that the claimed order is merely an obvious or arbitrary reordering of steps, and further asserts that the order of steps of coupling an amplification product to form a polymer conjugate, removing unreacted polymer, and shrinking/expanding the polymer conjugate is functionally tied to the claimed targeted shrinking/expanding of the amplification product. Applicant argues, based on this assertion, that the claimed order is a not a simple reordering of steps of Daugharthy, but rather a distinct process.
As discussed with regard to the arguments pertaining to the 35 U.S.C. 102 rejection above, the method of Daugharthy teaches a method that results in the targeted expansion of the amplification product. Therefore, the assertion that the order is functionally tied to the claimed targeted shrinking or expansion of the amplification product is not evidence of a new or unexpected result. Per MPEP §2144.04(IV)(C): “In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results)”. As Daugharthy teaches all of the respective steps of claim 1 (see 35 U.S.C. 103 rejection above) and achieves the same result of targeted expansion of the amplification product, the claimed order is a simple reordering/claim for a specified order, this argument is not persuasive. Sufficient showing that either Daugharthy does not teach one of the steps of the claim or that the specific ordering of the steps is critical and achieves new or unexpected results would be sufficient to overcome this rejection.
On pages 12-13, Applicant also argues that claims 3 and 29 are allowable for the same reasons as claim 1 upon which they depend. This is not persuasive, see discussion regarding the rejection of claim 1 above.
Applicant’s arguments, see pages 13-15, filed 22 June 2026, with respect to the rejection of claims 15, 32, 94, 95, 109, and 100 under 35 U.S.C. 103 as unpatentable over Daugharthy in view of Church have been fully considered but they are not persuasive.
On pages 13-15, Applicant argues that Daugharthy fails to teach the method of claim 1 as discussed previously or the corresponding limitations of claim 109 (from which claim 110 depends), and argues that Church does not cure these deficiencies of Daugharthy. As discussed with regard to claim 1 above, Examiner is not persuaded that the alleged deficiencies are present in Daugharthy, so the argument that Church does not cure these alleged deficiencies is not persuasive.
On pages 15 and 16, Applicant similarly argues regarding the rejections of claims 33 and 52 that the additional references of Ishidao, Asano, and Dewal fail to cure the alleged deficiencies of Daugharthy or Daugharthy and Church. As discussed with regard to claim 1 above, Examiner is not persuaded that the alleged deficiencies are present in Daugharthy or the combination of Daugharthy and Church, so these arguments are not persuasive.
Conclusion
Claims 1, 3, 5, 11, 15, 22, 28-29, 32-33, 45, 52-53, 79-80, 85, 94-95, and 109-110 are rejected. Claims 2, 4, 6-10, 12-14, 16-21, 23-27, 30-31, 34-44, 46-51, 54-78, 81-84, 86-93, and 96-108 are canceled.
Applicant's amendment necessitated the new ground(s) of rejection and modifications to rejections 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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/JEFFREY BELLAH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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