Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
The Examiner for this Application has changed. Please direct all future correspondence to Katriel Kasayan, AU 1634. Additional contact information can be found at the end of this paper.
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 in response to papers filed on September 3, 2025. Pursuant to amendments filed on July 17, 2025, claims 1-4 and 6-20 are currently pending. Applicants’ election without traverse of Group I, e.g. claims 1-4 and 6-12, in response to the restriction election filed June 9, 2023, has been previously acknowledged. Claims 13-20 were previously withdrawn from consideration pursuant to 37 CFR 1.142(b), as being drawn to non-elected invention, there being no allowable generic or linking claim. The restriction requirement is final.
Claim 5 has been previously canceled. Claim 1 has been amended in the amendments filed on September 3, 2025.
Therefore, claims 1-4 and 6-12 are currently under examination to which the following grounds of rejection are applicable.
Priority
The instant application claims priority to US Provisional Application 63/020,499 filed May 5, 2020.
Therefore, the earliest effective filing date for the instant application is May, 5, 2020.
Response to Arguments
Maintained Rejections in Response to Applicants’ amendments and arguments:
Claim Rejections - 35 USC § 112
Claims 1-4 and 6-12 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a New Matter rejection.
The limitations "(b) a thermal or radical initiator, wherein the initiator : hydrogel monomer molar ratio is in the range of 1:10,000 to 1 :20,000, and (c) a crosslinker, wherein the crosslinker : hydrogel monomer molar ratio is between 1: 170,000 to 1 :270,000" as recited in amended claim 1 and "the thermal initiator : hydrogel monomer molar ratio is 1 :14,000, and/or (b) the crosslinker : hydrogel monomer molar ratio is 1 :220,000" as recited in amended claim 4 are not supported by the original disclosure or claim as filed.
Applicant's arguments, filed 18 December 2024, asserts that no new matter has been added. However, the specification as filed does not provide sufficient written description of the above-mentioned limitations. The specification does not provide sufficient support for the recited molar ratio. The specification only discloses concentrations of the hydrogel monomer in weight/volume followed by ratios of the initiator to hydrogel monomer and crosslinker to hydrogel monomer, without mention of a molar ratio (Instant Specification Pg. 3, lines 3-18; Pg. 13, lines 10-20; Pg. 13, line 27-Pg. 14, line 4). The instant claims now recite "(b) a thermal or radical initiator, wherein the initiator : hydrogel monomer molar ratio is in the range of 1:10,000 to 1 :20,000, and (c) a crosslinker, wherein the crosslinker : hydrogel monomer molar ratio is between 1: 170,000 to 1 :270,000" and "the thermal initiator : hydrogel monomer molar ratio is 1 :14,000, and/or (b) the crosslinker : hydrogel monomer molar ratio is 1 :220,000", which were not clearly disclosed in the specification. Therefore, the claims represent a departure from the specification and claims originally filed.
Such limitations recited in the present claims, which did not appear in the specification, as filed, introduce new concepts and does not satisfy the description requirement of the first paragraph of 35 U.S.C. 112.
Response to Applicant arguments as they apply to the rejection of the claims under 35 U.S.C. 112(a), New Matter
Beginning on page 7 of remarks filed on September 3, 2025, Applicants essentially argue the following:
The skilled person would understand the claimed ratio is a molar ratio as it is the only reasonable interpretation in view of the specification as a whole and the claims that depend from claim 1.
“In the Example at paragraph [0112] of the published application, an ELAST solution is
disclosed as having: 30% (wt/vol) AAm, a hydrogel monomer; 0.0003% (wt/vol) MBAA, crosslinker; and 0.01 % (wt/vol) V A-044, an initiator. In this solution, the molar ratio of the crosslinker and hydrogel monomer is 1:~216,897”
Applicant's arguments have been fully considered but they are not persuasive for the following reason(s):
Regarding 1) and 2), Applicant argues that the Specification could have been converted to molar ratios that result within the claimed ranges. However, the instant claims recite limitations involving relative amounts of the hydrogel monomer, initiator and crosslinker in terms of weight/volume concentrations. A weight/volume concentration or ratio is not equivalent to a molar ratio, rather the former expresses an amount of material based on mass and the solution volume, whereas molar ratio is determined on the amount of moles and therefore additionally depends upon the respective molecular weights of the components. A person of ordinary skill would have to perform undue experimentation to calculate the correct molar ratio based on the full scope of the claims. Furthermore, with respect to applicants' argument that, "an ELAST solution is disclosed [VA-044 stock] as having: 30% (wt/vol) AAm, a hydrogel monomer; 0.0003% (wt/vol) MBAA, a crosslinker; and 0.01 % (wt/vol) V A-044, an initiator” (para [0112] of the published application) ,is not found persuasive because it is noted that the features upon which applicant relies (i.e., percentages (wt/volume) useful according to the instant specification at para [0112] of the published application are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26USPQ2d 1057 (Fed. Cir. 1993). This is the case here. The claims do not recite the ratio of (wt/vol) taught in the specification.
Claim Rejections - 35 USC § 103
Claims 1-4 and 6-12 remain rejected under 35 U.S.C. 103 as being unpatentable over Ku (Ku, T., et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues, 2016, Nature Biotechnology, 34(9):973-81; 11 September 2023 IDS Document; previously cited) in view of Chen (US20180119219A 1; previously cited) as evidenced by Chung (Chung, K., et al. Chung Lab CLARITY Protocol, 2016; previously cited).
Regarding claims 1-3, 6 and 8, Ku teaches a method, called magnified analysis of the proteome (MAP), [that] linearly expands entire organs four-fold while preserving their overall architecture and three-dimensional proteome organization. (Ku Abstract lines 4-6). MAP preserves both the three-dimensional (3D) proteome content and organization and organ-wide cellular connectivity within an intact tissue-hydrogel hybrid while rendering it reversibly size adjustable up to four- to five-fold for multiresolution imaging (Ku Pg. 3, [1], lines 3-6), readding on “thereby producing a tissue-hydrogel capable of reversible non-uniform shape and size transformation”. Ku et al., teaches tissue expansion was reversible and tunable using buffers with different salt concentrations and osmolarities (Ku Pg. 3, [4], lines 2-3). Ku further teaches that mice were first washed transcardially with a mixture of 2-5% AA, 0-0.05% BA, 0-0.8% sodium acrylate (SA), and PBS, followed by perfusion with a mixture of 4% PFA (paraformaldehyde), 30% AA (acrylamide), 0.05-0.1 % BA (N,N'-methylenebisacrylamide), 10% SA, 0.1% VA-044 or V-50, and PBS. The brain [as recited in claim 8] and other organs (heart, lung, liver, intestine, kidney, and spinal cord) were harvested and incubated in 20-40 ml of the same fixative solution (4% PFA (paraformaldehyde), 30% AA (acrylamide) [as recited in claims 1(a), 2, 3(a) and 6(a)], 0.05-0.1% BA (N,N'-methylenebisacrylamide), 10% SA, 0.1% VA-044 or V-50, and PBS) at 4°C for 2-3 days and then for 1-3 days at room temperature (RT) with gentle shaking to ensure uniform chemical diffusion and reaction throughout the sample. Following the diffusion and fixation steps, hydrogel-tissue hybridization was performed in situ by incubating the tissues using EasyGel (LifeCanvas Technologies, Seoul, South Korea) with nitrogen gas at 45°C for 2 h (Ku Pg. 8, 2nd passage, lines 5-17). Hydrogel-tissue hybridization by incubating tissues with nitrogen gas is through removal of oxygen as evidenced by Chung (Chung Pg. 7, #7: Removal of oxygen is necessary for hydrogel-tissue hybridization because oxygen radicals may terminate the polymerization reaction. #8: Connect nitrogen gas and a vacuum pump to the desiccator via the 3-way stopcock. Open flow in all three directions and turn on the nitrogen gas. Allow the gas to flow for about 5 seconds. This step is necessary to flush oxygen from all the tubing in the system).
Ku does not teach the thermal initiator: hydrogel monomer ratio is 1:10,000 - 1 :20:000 or the crosslinker: hydrogel monomer ratio is 1 :170,000 - 1 :270,000 as recited in claim 1 (b) and (c ), the thermal initiator: hydrogel monomer ratio is 1 :14,000 or the crosslinker: hydrogel monomer ratio is 1 :220,000 as recited in claim 4; or wherein (b) VA-044 is 0.01 % wt/vol, and (c) MBAA is 0.0003% wt/vol as recited in claim 6.
However, Ku teaches the key to MAP is to prevent intra-and inter-protein crosslinking during the hydrogel-tissue hybridization step and then to dissociate and denature proteins to allow natural expansion of the hybrid. [It was] hypothesized that a high concentration of acrylamide (AA) monomers might effectively prevent protein crosslinking by quenching reactive methylols formed by the protein-formaldehyde reaction. If the AA monomer concentration is low, the reactive methylols would react with amide groups within the same protein or adjacent proteins to form methylene bridges. Such intra- and inter-protein crosslinking would prevent dissociation of protein complexes and limit subsequent tissue expansion. With increased AA concentration, methylols might preferentially react with excess AA monomers, effectively reducing inter-protein crosslinking while maximally tethering individual proteins to an expandable hydrogel mesh (Ku Pg. 3, [21). Phantoms fixed in higher concentrations of AA showed higher degrees of expansion in water (Ku Pg. 3, [3], lines 5-6).
The person of ordinary skill in the art would have found it obvious to optimize the concentration of the hydrogel monomer/acrylamide through routine experimentation to obtain a ratio of low ratio of thermal initiator or crosslinker to a higher amount of hydrogel monomer as recited in claims 1, 4 and 6, because Ku discloses tissues in higher concentrations of acrylamide showed higher degrees of expansion in water by reducing by quenching reactive methylols formed by the protein-formaldehyde reaction and thus effectively reducing inter-protein crosslinking (Ku Pg. 3, [3], lines 5-6).
Chen discloses techniques for expansion microscopy (Chen Abstract, lines 3-4) in which chemically fixed and permeabilized biological specimens are embedded in a swellable gel material, subjected to a treatment to disrupt native biological networks, and then expanded (Chen [86], lines 1-4). Chen further discloses gel recipes, varying in monomer, cross-linker, initiator, accelerator, inhibitor and other additives can be used to tune the hydrogel properties such as expansion factor, chemical environment, and mechanical properties. These monomers include, acrylamide variants such as dimethylacrylamide, hydroxymethylacrylamide and acrylamide. Other free radical initiators such as VA-044 or UV activated (Chen [921).
The person of ordinary skill in the art would have found it obvious to optimize the concentration of crosslinker/MBAA and thermal initiator/VA-044 as recited in claims 1, 4 and 6, because Chen teaches the crosslinker and initiator can be used to tune hydrogel properties, such as expansion factor, chemical environment and mechanical properties (Chen [92]).
Regarding claim 7, Ku further teaches reconstruction of individual neurons requires
labeling and imaging of thick brain tissues because nerve fibers can extend across a large
volume. To test whether MAP is applicable to large-scale brain tissues, we expanded a 1-mmthick
mouse brain block (5-mm-thick after expansion) (Ku Pg. 6, lmmunolabeling and imaging
of mm-thick tissues, lines 1-3). Ku teaches 1-and 2-mm-thick mouse brain coronal slices were prepared by "General MAP protocol", and expanded (Ku Pg. 10, Large tissue staining, line 1). Ku also teaches a four-fold linear expansion of a whole mouse brain tissue (Ku Pg. 3, [4], line 1; Pg. 15-16: Fig. 1f). Figure 1f depicts a whole mouse brain with approximate dimensions of 45 mm x 60 mm (scale bar is 10 mm).
Ku does not teach the tissue fragment has lateral dimensions of 6 cm by 8 cm.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use a tissue fragment with larger dimensions of 6 cm by 8 cm in Ku's method of making a tissue-hydrogel hybrid, because Ku's method allows preservation and magnification of whole organs by preventing intra- and inter-protein crosslinking, denaturing, and dissociating of protein complexes to allow natural expansion of hydrogel-tissue hybrids (Ku Pg. 6, Discussion, lines 4-7).
Regarding claim 9, Chen discloses techniques for expansion microscopy (Chen Abstract, lines 3-4) in which a sample, such as tissue, can be permeabilized. A permeabilized sample, or tissue, can be infused with monomers or precursors of a swellable material and then causing the monomers or precursors to undergo polymerization within the sample to form the swellable material (Chen [54], lines 10-15). Chen discloses an embodiment in which the sample is embedded in a swellable or unswellable hydrogel following permeabilization of the fixed biological sample [as recited in claim 9] (Chen [561). Monomers include acrylamides (Chen [72], lines 13-15). The biological sample can be treated with a detergent prior to being contacted with one or more swellable material precursors. The use of a detergent improves the wettability of the sample or disrupt the sample to allow the one or more swellable monomer precursors to permeate throughout sample (Chen [771).
Regarding claims 10b and 12b, Chen further discloses the [brain tissue] slice was immediately fixed with 4% Formaldehyde in 1 xPBS for 12 minutes, washed three times with 1 xPBS, briefly treated ( <2 minutes) with 4% Sodium dodecyl sulfate in 1 xPBS, washed three times in 1xPBS, and then stored overnight in 70% ethanol@ 4°C (Chen [2171).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ku's method of making a tissue-hydrogel hybrid by permeabilizing the tissue prior to contacting the tissue with hydrogel monomers, because Chen teaches the use of a detergent can improve the wettability of the sample or disrupt the sample to allow the one or more swellable monomer precursors to permeate throughout sample (Chen [77]). One of ordinary skill in the art would have been capable of applying a known technique of fixing a tissue sample in formaldehyde prior to contacting the sample with hydrogel monomers in a known method of making a tissue-hydrogel and the results would have been predictable to one of ordinary skill in the art.
Regarding claim 11, Ku teaches hydrogel-embedded tissues were incubated overnight in a solution of 200 mM SOS at 37°C with gentle shaking. The samples were then incubated at 70°C for 0-50h and 95°C for 1-24 h depending on their size (Ku Pg. 8, ii. Tissue denaturation), but does not teach wherein the tissue fragment is permeabilized prior to step (1) by contacting the tissue fragment with a detergent at a concentration greater than about 0.5% wt/vol at a temperature of 20-80°C for about 1-10 days.
Chen discloses contacting the tissue fragment with a detergent (SOS) at a concentration greater than about 0.5% wt/vol (Chen [2171).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ku's method of making a tissue-hydrogel hybrid by permeabilizing the tissue prior to contacting the tissue with hydrogel monomers, because Chen teaches the use of a detergent can improve the wettability of the sample or disrupt the sample to allow the one or more swellable monomer precursors to permeate throughout sample (Chen [77]). Routine optimization of the temperature and duration in the step of contacting the tissue fragment with detergent would have led to the claimed range of 20-80°C for about 1-10 days, because Ku discloses incubating samples at 70°C for 0-50 h (Ku Pg. 8, ii. Tissue denaturation). One of ordinary skill in the art would have found it obvious to optimize within the range taught by Ku, because Ku teaches the incubation time and temperature is dependent on the sample size.
Response to Applicant arguments as they apply to the rejection of the claims under 35 U.S.C. 103
Beginning on page 7 of remarks filed on September 3, 2025, Applicants essentially argue the following:
There is no motivation to combine or modify Ku with Chen, and no reasonable expectation of success in doing so, and that Chen teaches away from Ku
The combination of Ku and Chen does not yield the claimed method of producing a tissue-hydrogel hybrid capable of reversible non-uniform shape and size transformation
The claimed invention is associated with unexpected results
Applicant's arguments have been fully considered but they are not persuasive for the following reason(s):
Regarding 1), although the examiner acknowledges Ku’s criticism on expansion microscopy (ExM), specifically on the loss of proteins during the step of protease digestion, Chen is not relied upon for its methods of expanding tissue through hydrogel interactions. Rather, Chen is relied upon for the teaching that known hydrogel formulation variables, such as monomers, crosslinkers and initiators, may be varied and fine-tuned based on influential considerations of the design such as expansion factor, chemical environment and mechanical properties (para 0091-0092). Therefore, the proposed combination does not require incorporating Chen’s protease treatment into Ku, and the criticism of protease digestion would not have discouraged the specific modification of the ratios of monomers, crosslinkers and initiators.
The Court has stated that generally such differences amount to mere optimization and will not support patentability unless there is evidence indicating the claimed feature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.). For more recent cases applying this principle, see Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997). In KSR International Co. v. Teleflex Inc., 550 U.S. 398 (2007), the Supreme Court held that "obvious to try" was a valid rationale for an obviousness finding, for example, when there is a "design need" or "market demand" and there are a "finite number" of solutions. 550 U.S. at 421.
MPEP § 2144 sets forth Applicant' s burden for rebuttal of a prima facie case of obviousness based upon routine optimization. Applicant must provide either a showing that the particular amount or range recited within the claims is critical; and/or a showing that the prior art reference teaches away from the claimed amount.
Regarding 2), the Examiner acknowledges that Ku teaches the magnified analysis of the proteome (MAP), linearly expands, as recited in the Abstract (pp. 97). However, the claim does not recite objective criteria defining the boundary between the non-uniform transformation and other deformation of a tissue-hydrogel hybrid. Thus, it has been held that the recitation that an element is “capable of” performing a function is not a positive limitation but only requires the ability to so perform. It does not constitute a limitation in any patentable sense. In re Hutchinson, 69 USPQ 138. Furthermore, Ku discloses that if acrylamide monomer concentration is low, causing crosslinking which subsequently influences tissue expansion (pp. 974 col 2 bridging into pp. 975 col 1). Conversely, a high concentration of acrylamide monomers might effectively prevent protein crosslinking by quenching reactive methylols formed by the protein-formaldehyde reaction. Thus, Ku reveals that acrylamide concentrations influence tissue expansion (pp. 976, col 1, para 1), and that tissue expansion was reversable by using buffers with different salt concentrations and osmolarities (pp. 976, col 1, para 2). Therefore, one of ordinary skill in the art would have recognized that acrylamide concentrations, as discussed in Ku, and significantly low relative amounts of hydrogel components, e.g., varying in monomer, cross-linker, initiator, accelerator, inhibitor and other additives, as discussed in Chen (para 0092), present as result effective variables that a skilled artisan would routinely optimize based on influential considerations in the design of the reversible tissue-hydrogel, such as the non-uniform expansion of the hydrogel, as Ku is silent on whether the uniform expansion with minimal deformities is achieved for all concentrations of the tissue-hydrogel hybrid.
Regarding 3), Applicant assets that the claimed method produces unexpected elasticity and stretchability, as discussed in the previous Remarks filed March 29, 2024. However, Ku already demonstrates that tissue-hydrogel hybrids could be expanded four-fold while preserving their overall architecture (pp. 973, Abstract), which is not unpredictable in the art. Furthermore, Ku teaches that altering the acrylamide concentration and osmolarity can be detrimental to tissue expansion (pp. 976, col 1, para 1-2). Thus, since Ku already discloses that the modification of the hydrogel composition can be tuned for tissue expansion (pp. 976 col 1 para 2), the elasticity does not constitute an unexpected result.
Moreover, page 22 lines 27-30 of the instant Specification reveal an ELAST formulation of 30% acrylamide (Aam), 0.01% VA-044, and 0.0003% of MBAA. The proclaimed unexpected results must be considered to the breadth of claim 1, which broadly encompasses hydrogel monomer concentrations between the range of 20%-70%, ratios of the crosslinker to monomers, etc. As such, it has not been established that the unexpected properties are reasonably corresponding with the full scope of the claims.
Double Patenting
Claims 1-12 remain rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 6, 7 and 10 of U.S. Patent No.11397140 (Chung) in view of Ku (Ku, T., et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues, 2016, Nature Biotechnology, 34(9):973-81; 11 September 2023 IDS Document) and Chen (US20180119219A1) as evidenced by Chung (Chung, K., et al. Chung Lab CLARITY Protocol, 2016, https ://static 1 .squarespace .com/static/561 beb9ae4b0edc5a 7075cf2/t/57ba20d69f7 456e36f2f6c e3/1504722155038/Chung+lab_CLARITY +protocol_08192016.pdf).
Patent claim 1 recites a method for preserving a tissue comprising: perfusing or incubating a tissue with hydrogel subunits, inducing polymerization of the hydrogel subunits under conditions comprising a concentration of hydrogel subunits that ranges from 25-80% weight/volume, and denaturing and/or dissociating biomolecules in the tissue, thereby forming a size-adjustable tissue-hydrogel hybrid, wherein the hydrogel subunits are acrylamide monomers. Patent claim 2 recites wherein the tissue is perfused or incubated under conditions that minimize inter- or intra-tissue binding. Patent claim 6 recites the method of claim 1, wherein denaturing and/or dissociating biomolecules comprises contacting the tissue with a detergent, incubating at high temperature, mechanical dissociation, or sonication. Patent claim 7 recites wherein the tissue-hydrogel hybrid is reversibly and proportionally size-adjustable in three dimensions. Patent claim 10 recites wherein the tissue has been perfused and fixed with fixative prior to being incubated with hydrogel subunits.
The patent claims do not recite contacting a tissue fragment, at a temperature of about 0-4°C for about 1-10 days, with thermal initiator: hydrogel monomer ratio is 1:10,000 - 1 :20:000 and the crosslinker: hydrogel monomer ratio is 1 :170,000 - 1 :270,000; and increasing the temperature to a temperature in the range of about 1-30°C for about 2 hours to 1 day, and removing oxygen as recited in instant claim 1; the thermal initiator is VA-044 and the crosslinker is N,N'-methylenebisacrylamide (MBAA) in instant claim 2; the thermal initiator: hydrogel monomer ratio is 1 :14,000 and/or the crosslinker: hydrogel monomer ratio is 1 :220,000 as recited in instant claim 4; VA-044 and is present at 0.01% wt/vol, and MBAA and is present at 0.0003% wt/vol as recited in instant claim 6; the tissue fragment about 2-5 mm thick with lateral dimensions of 6 cm by 8 cm as recited in instant claim 7; wherein the tissue fragment is a human tissue fragment, optionally a heart, colon or brain tissue fragment, optionally a cerebral organoid as recited in instant claim 8; wherein the tissue is contacted with formaldehyde before step (1) as recited in instant claim 10; wherein the tissue fragment is permeabilized prior to step (1) by contacting the tissue fragment with a detergent at a concentration of greater than about 0.5% wt/vol at a temperature of 20-80°C for about 1-10 days as recited in instant claim 11; and the detergent is a non-ionic detergent and/or sodium dodecyl sulfate (SOS) as recited in instant claim 12.
Ku teaches a method, called magnified analysis of the proteome (MAP), [that] linearly expands entire organs four-fold while preserving their overall architecture and three-dimensional proteome organization. (Ku Abstract lines 4-6). MAP preserves both the three-dimensional (3D) proteome content and organization and organ-wide cellular connectivity within an intact tissue-hydrogel hybrid while rendering it reversibly size-adjustable up to four- to five-fold for multiresolution imaging (Ku Pg. 3, [1], lines 3-6). Tissue expansion was reversible and tunable using buffers with different salt concentrations and osmolarities (Ku Pg. 3, [4], lines 2-3).
Regarding instant claims 1-2 and 8, Ku teaches mice were first washed transcardially with a mixture of 2-5% AA, 0-0.05% BA, 0-0.8% sodium acrylate (SA), and PBS, followed by perfusion with a mixture of 4% PFA (paraformaldehyde), 30% AA (acrylamide), 0.05-0.1 % BA (N,N'-methylenebisacrylamide), 10% SA, 0.1% VA-044 or V-50, and PBS. The brain [as recited in claim 8] and other organs (heart, lung, liver, intestine, kidney, and spinal cord) were harvested and incubated in 20-40 ml of the same fixative solution (4% PFA (paraformaldehyde), 30% AA (acrylamide), 0.05-0.1% BA (N,N'-methylenebisacrylamide), 10% SA, 0.1% VA-044 or V-50, and PBS) at 4°C for 2-3 days and then for 1-3 days at room temperature (RT) with gentle shaking to ensure uniform chemical diffusion and reaction throughout the sample. Following the diffusion and fixation steps, hydrogel-tissue hybridization was performed in situ by incubating the tissues using Easy-Gel (LifeCanvas Technologies, Seoul, South Korea) with nitrogen gas at 45°C for 2 h (Ku Pg. 8, [2], lines 5-17). Hydrogeltissue hybridization by incubating tissues with nitrogen gas is through removal of oxygen as evidenced by Chung (Chung Pg. 7, #7: Removal of oxygen is necessary for hydrogel-tissue hybridization because oxygen radicals may terminate the polymerization reaction. #8: Connect nitrogen gas and a vacuum pump to the desiccator via the 3-way stopcock. Open flow in all three directions and turn on the nitrogen gas. Allow the gas to flow for about 5 seconds. This step is necessary to flush oxygen from all the tubing in the system).
Regarding instant claim 4, Ku teaches the key to MAP is to prevent intra-and interprotein crosslinking during the hydrogel-tissue hybridization step and then to dissociate an denature proteins to allow natural expansion of the hybrid. [It was] hypothesized that a high concentration of acrylamide (AA) monomers might effectively prevent protein crosslinking by quenching reactive methylols formed by the protein-formaldehyde reaction. If the AA monomer concentration is low, the reactive methylols would react with amide groups within the same protein or adjacent proteins to form methylene bridges. Such intra- and inter-protein crosslinking would prevent dissociation of protein complexes and limit subsequent tissue expansion. With increased AA concentration, methylols might preferentially react with excess AA monomers, effectively reducing inter-protein crosslinking while maximally tethering individual proteins to an expandable hydrogel mesh (Ku Pg. 3, [21). Phantoms fixed in higher concentrations of AA showed higher degrees of expansion in water (Ku Pg. 3, [3], lines 5-6).
Regarding instant claim 7, Ku further teaches reconstruction of individual neurons requires labeling and imaging of thick brain tissues because nerve fibers can extend across a large volume. To test whether MAP is applicable to large-scale brain tissues, we expanded a 1- mm-thick mouse brain block (5-mm-thick after expansion) (Ku Pg. 6, lmmunolabeling and imaging of mm-thick tissues, lines 1-3). Ku teaches 1-and 2-mm-thick mouse brain coronal slices were prepared by "General MAP protocol", and expanded (Ku Pg. 10, Large tissue staining, line 1 ). Ku also teaches a four-fold linear expansion of a whole mouse brain tissue (Ku Pg. 3, [4], line 1; Pg. 15-16: Fig. 1f). Figure 1f depicts a whole mouse brain with approximate dimensions of 45 mm x 60 mm (scale bar is 10 mm).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the step of incubating a tissue with hydrogel subunits in the patent method by further contacting the tissue with a thermal initiator/VA-044 and a crosslinker/MBAA at 0-4° for about 1-10 days and then increasing the temperature to 1-30° for about 2 hours to 1 days and removing oxygen as disclosed by Ku. One of ordinary skill in the art would have been capable of applying this known technique to a known method of producing a hydrogel-tissue hybrid and the results would have been predictable to one of ordinary skill in the art.
The person of ordinary skill in the art would have found it obvious to optimize the concentration of the hydrogel monomer/acrylamide through routine experimentation to obtain a ratio of thermal initiator or crosslinker to a higher amount of hydrogel monomer as recited in instant claims 1 and 4, because Ku discloses tissues in higher concentrations of acrylamide showed higher degrees of expansion in water (Ku Pg. 3, [3], lines 5-6).
Pertaining to instant claim 7, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use a tissue fragment with larger dimensions of 6 cm by 8 cm in Ku's method of making a tissue-hydrogel hybrid, because Ku's method allows preservation and magnification of whole organs by preventing intra- and interprotein crosslinking, denaturing, and dissociating of protein complexes to allow natural expansion of hydrogel-tissue hybrids (Ku Pg. 6, Discussion, lines 4-7).
Chen discloses techniques for expansion microscopy (Chen Abstract, lines 3-4) in which chemically fixed and permeabilized biological specimens are embedded in a swellable gel material, subjected to a treatment to disrupt native biological networks, and then expanded (Chen [86], lines 1-4). Chen further discloses gel recipes, varying in monomer, cross-linker, initiator, accelerator, inhibitor and other additives can be used to tune the hydrogel properties such as expansion factor, chemical environment, and mechanical properties. These monomers include, acrylamide variants such as dimethylacrylamide, hydroxymethylacrylamide and acrylamide. Other free radical initiators such as VA-044 or UV activated (Chen [921).
One of ordinary skill in the art would have found it obvious to optimize the concentration of crosslinker/MBAA and thermal initiator/VA-044 as recited in instant claims 1, 4 and 6, because Chen teaches the crosslinker and initiator can be used to tune hydrogel properties, such as expansion factor, chemical environment and mechanical properties (Chen [92]).
Regarding instant claims 10b and 12b, Chen further discloses the [brain tissue] slice was immediately fixed with 4% Formaldehyde in 1 xPBS for 12 minutes, washed three times with 1xPBS, briefly treated (<2 minutes) with 4% Sodium dodecyl sulfate in 1xPBS, washed three times in 1xPBS, and then stored overnight in 70% ethanol@ 4°C (Chen [2171).
One of ordinary skill in the art would have been capable of applying a known technique of fixing a tissue sample in formaldehyde prior to contacting the sample with hydrogel monomers in a known method of making a tissue-hydrogel and the results would have been predictable to one of ordinary skill in the art.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the patent claim's method of making a tissue-hydrogel hybrid by permeabilizing the tissue with SOS prior to contacting the tissue with hydrogel monomers, because Chen teaches the use of a detergent can improve the wettability of the sample or disrupt the sample to allow the one or more swellable monomer precursors to permeate throughout sample (Chen [77]).
Regarding instant claim 11, Ku teaches hydrogel-embedded tissues were incubated overnight in a solution of 200 mM SOS at 37°C with gentle shaking. The samples were then incubated at 70°C for 0-50h and 95°C for 1-24 h depending on their size (Ku Pg. 8, ii. Tissue denaturation), but does not teach wherein the tissue fragment is permeabilized prior to step (1) by contacting the tissue fragment with a detergent at a concentration greater than about 0.5% wt/vol at a temperature of 20-80°C for about 1-1 O days as recited in claim 11.
Routine optimization of the temperature and duration in the step of contacting the tissue fragment with detergent would have led to the claimed range of 20-80°C for about 1-10 days, because Ku discloses incubating samples at 70°C for 0-50 h (Ku Pg. 8, ii. Tissue denaturation). The personal of ordinary skill in the art would have found it obvious to optimize within the range taught by Ku, because Ku teaches the incubation time and temperature is dependent on the sample size.
Response to Applicant arguments as they apply to the rejection of the claims under Double Patenting
Applicant requests that the rejection be held in abeyance until allowable subject matter is identified. A complete response to a nonstatutory double patenting (NSDP) rejection is either a reply by applicant showing that the claims subject to the rejection are patentably distinct from the reference claims, or the filing of a terminal disclaimer in accordance with 37 CFR 1.321 in the pending application(s) with a reply to the Office action (see MPEP § 1490 for a discussion of terminal disclaimers). Such a response is required even when the nonstatutory double patenting rejection is provisional. As filing a terminal disclaimer or filing a showing that the claims subject to the rejection are patentably distinct from the reference application’s claims, is necessary for further consideration of the rejection of the claims, such a filing should not be held in abeyance. Only compliance with objections or requirements as to form not necessary for further consideration of the claims may be held in abeyance until allowable subject matter is indicated. Replies with an omission should be treated as provided in MPEP § 714.03. Therefore, an application must not be allowed unless the required compliant terminal disclaimer(s) is/are filed and/or the withdrawal of the nonstatutory double patenting rejection(s) is made of record by the examiner. See MPEP § 804(I)(B)(1).
Conclusion
No claims allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Katriel B Kasayan whose telephone number is (571)272-1402. The examiner can normally be reached 10-4p.
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/KATRIEL BARCELLANO KASAYAN/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634
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