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 .
Claim Status and Formal Matters
This action is in response to papers filed 11/21/2025.
Claims 170, 175, 178, 182 have been amended.
Claims 170-176, 178-189 are pending.
The instant response is non-compliant with 37 CFR 1.121 as claims 177 has been canceled, but does the limitations of the claims. Further the response identified claims 189 as withdrawn, but the claim is not withdrawn. However, to promote compact prosecution and customer service the instant response will be examined. Future amendments which are non-compliant with 37 CFR 1.121 may not be entered or examined.
Applicant’s election without traverse of Group I, wherein clearing proteins and/or lipids from the sample comprises "degrading proteins"; * wherein clearing RNA and/or DNA and/or the extracellular matrix comprises "degrading extracellular matrix"; * wherein the acrydite portion is bound to the "5' end"; * wherein clearing comprises exposing the gel to a "proteinase"; * wherein imaging comprises using "epi-fluorescence microscopy" in the reply filed on 4/14/2025is acknowledged.
Claims 172, 174, 181, 186-188 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention/species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 4/14/2025.
Claims 170-171, 173, 175-176, 178-180, 182-185, and 189 are being examined.
The sequence listing issue has been withdrawn in view of the amendment to the specification to identify the sequences of table 1 by SEQ ID NO.
The previous objection to the claims has been withdrawn in view of the amendment to the claims.
The previous objection to the specification has been withdrawn in view of the amendment to the claims.
The previous new matter rejection has been withdrawn in view of the amendment to the claims to delete the language not supported by the specification.
The ODP rejection has been withdrawn in view of the amendment.
Priority
The instant application was filed 05/01/2023 and is a continuation of 16347874 , filed 05/07/2019, which is a national stage entry of PCT/US2017/060570 with an international filing date: 11/08/2017 and claims priority from provisional application 62419033 , filed 11/08/2016.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/21/2025 is being considered by the examiner.
It is noted that the IDS contain office actions related to foreign applications, but do not provide a context or specific claims or references that are discussed in the actions. These have been reviewed for what is provided within them, but the relevant application material such as specification, claims and cited references have not been used for the review.
Drawings
The specification teaches, FIGS. 3A-3E illustrate a reduction of background in multiple color imaging, in yet another embodiment of the invention;.” Thus the drawings appear to be in color. “Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Response to Arguments
The response traverses the rejection asserting the drawings are not color. This argument has been thoroughly reviewed but is not considered persuasive as the specification states, “, FIGS. 3A-3E illustrate a reduction of background in multiple color imaging, in yet another embodiment of the invention.”
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 170-171, 173, 175-180, 182-185, and 189 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 170 is indefinite because it lacks a positive active step relating back to the preamble. The preamble recites a method for gel embedding a sample, however the last positive active step is drawn to determining binding of the primary nucleic acid probes to the nucleic acid targets by imaging the polyacrylamide gel wherein the primary probes are contacted with a plurality of secondary nucleic acid probes comprising a fluorescent dye and a recognition sequence that hybridizes to the one or more read sequence of the primary probe.. Therefore it is unclear as to whether the method is drawn to gel embedding a sample or determining binding of the primary nucleic acid probes to the nucleic acid targets by imaging the polyacrylamide gel wherein the primary probes are contacted with a plurality of secondary nucleic acid probes comprising a fluorescent dye and a recognition sequence that hybridizes to the one or more read sequence of the primary probe.
Response to Arguments
The response traverses the previous rejection in view of the amendment. The amendments have overcome the prior rejections, but introduced new issues.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 170-171, 173, 175-176, 178, 182-185 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen (WO2015127183), Trafton (MIT news 1/15/2015) Thomsen (RNA (2005) volume 11, pages 1745-1748), Alon (2015 SCSB FALL POSTER SESSION ABSTRACTS)
Chen teaches, “The present invention relates to an enlarged sample of interest for microscopy and methods for enlarging a sample of interest and the optical imaging of a sample of interest with resolution better than the classical microscopy diffraction limit, by synthesizing a swellable polymer network within a sample, it can be physically expanded, resulting in physical magnification.”
Chen teaches, “[0008] In an embodiment of this concept, the composition comprises a polyelectrolyte hydrogel ( or the components thereof), which can swell macroscopically, for example, in low-salt water. The composition can comprise a tag or other feature of interest (for example, fluorescent dye molecules that have been delivered to the biological sample via antibody staining) which can be anchored (e.g., chemically) into the hydrogel before expansion. Following anchoring, the specimen is subjected to an enzymatic digestion ( or other digestion) to disrupt the underlying network of biological molecules, leaving the tags of interest (e.g., the fluorescent dye molecules) intact and anchored to the gel. In this way, the mechanical properties of the gel-biomolecule hybrid material are rendered more spatially uniform, allowing isotropic expansion with minimal artifacts.”
Chen teaches, “ [0026] In a preferred embodiment, the sample of interest can be labeled or tagged. Typically, the label or tag will bind chemically ( e.g., covalently, hydrogen bonding or ionic bonding) to the sample, or a component thereof. The tag can be selective for a specific target (e.g., a biomarker or class of molecule), as can be accomplished with a 20 antibody or other target specific binder. The tag preferably comprises a visible component, as is typical of a dye or fluorescent molecule. Contacting the sample of interest with a label or tag results in a "labeled sample of interest." A fluorescently labeled sample of interest, fix example, is a sample of interest labeled through techniques such as, but not limited to, immunofluorescence, immunohistochemical or immunocytochemical staining to assist in microscopic analysis. Thus, the label or tag is preferably chemically attached to the sample of interest, or a targeted component thereof. In a preferred embodiment, the label or tag, e.g. the antibody and/or fluorescent dye, further comprises a physical, biological, or chemical anchor or moiety that attaches or crosslinks the sample to the composition, hydrogel or other swellable material. The labeled sample may furthermore include more than one label. For example, each label can have a particular or distinguishable fluorescent property, e.g., distinguishable excitation and emission wavelengths. Further, each label can have a different target specific binder that is selective for a specific and distinguishable target in, or component of the sample.” Chen teaches an anchor moiety.
With regards to claim 170, Chen teaches tissue preparation (0040) including slices were incubated with DNA-labeled secondary antibodies in hybridization buffer at a concentration of approximately 10 ug/mL for 6-12 hours, then washed in slice blocking buffer as for primary. Specimens were incubated with dye-labeled DNA tertiaries in hybridization buffer at a concentration of 1 ng/uL for 6-1 hours, then washed in slice blocking buffer as for primary.” Chen further teaches hydrogel embedding (polymerization) to anchor the probes via the acrylate modified 5’ end of the probes (0041). Chen teaches digestion of DNA by digestion with proteinase K and expansion (0042). Chen teachings imaging (0043).
Thus Chen anticipates the active steps of the claims by exposing ta fixed sample to the oligonucleotide modified with 5’ acrylate moieties, anchoring (embedding) the target and modified oligonucleotide in the gel by polymerization, expanding, clearing the gel by use of a proteinase K and imaging by used of secondary nucleic acid probes.
Chen teaches polymerization is a separate step followed by swelling or expansion (0027-0032) and (0041-0042). Chen (wo) states, “[0035] In one embodiment, the addition of water allows for the embedded sample to expand 4x to 5x (e.g., 4.5x) or more its original size in 3-dimensions. Thus, the sample can be increased 100-fold or more in volume. This is because the polymer is embedded throughout the sample, therefore, as the polymer swells (grows) it expands the tissue as well. Thus, the tissue sample itself becomes bigger. Surprisingly, as the material swells isotopically, the anchored tags maintain their relative spatial relationship. [0036] The swollen material with the embedded sample of interest can be imaged on any optical microscope, allowing effective imaging of features below the classical diffraction limit. Since the resultant specimen is preferably transparent, custom microscopes capable of large volume, wide field of view, 3-D scanning may also be used in conjunction with the expanded sample.”
Chen does not specifically teach detection of mRNA or use of oligo dT or alternating oligo dT with locked dT.
However, Trafton teaches, “Before enlarging the tissue, the researchers first label the cell components or proteins that they want to examine “
Alon suggest expansion microscopy for RNA expression.
Further, Thomsen teaches, “The usage of locked nucleic acid (LNA)-modified oligonucleotide probes has been shown to significantly improve the sensitivity and specificity of microRNA detection (Valoczi et al. 2004; Wienholds et al. 2005). LNA oligonucleotides are a new class of bicyclic RNA analogs that exercise an unprecedented high affinity for their complementary DNA or RNA targets (Koshkin et al. 1998). By using a design in which several positions in a conventional DNA oligonucleotide were substituted by LNAs, the sensitivity in detecting mature miRNAs by Northern blotting was increased by at least one order of magnitude (Valoczi et al. 2004). More recently, LNA-modified DNA-oligonucleotides were also used as FISH-probes on whole-mount zebrafish embryos to detect the temporal and spatial expression pattern of 115 conserved vertebrate miRNAs (Wienholds et al. 2005)” (page 1745, 1st column-2nd column). Thomsen teaches in table 1 probes with LNA from 32% of the probe to 35 % of the probe including oligo-dT.
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use the oligo dT including locked dT of Thomsen in the art of as either the tag(or label) or alternative as a replacement for the antibody and in anchor probe in a gel. The artisan would be motivated to take advantage of the unprecedented affinity and/or sensitivity in the method and determine if gel expansion alters specificity or distortion. The artisan would be motivated to detect mRNA at the cellular levels. The artisan would have a reasonable expectation of success as the artisan is merely substituting one nucleic acid or detection reagent for known oligo dT with LNA.
With regards to claim 171, Chen teaches anchor to polyacrylamide gel (0026-0027).
With regards to claim 173 and 175 , Chen teaches proteinase K digestion with degrades proteins which encompasses the extracellular matrix. (0042)
With regards to claim 176, Trafton and Alon teaches detection of RNA (example 4)
With regards to claim 182-183, Chen teaches, “Proteinase K (New England Biolabs) was diluted to 200ug/mL in digestion buffer (50mM Tris pH8, lmM EDTA, 0.5% TritonXlO0, IM NaCl, 0.8M guanidine HCl)” (0042)
With regards to claim 184, Chen teaches, “[0032] In a preferred embodiment, the sample (e.g., a labeled sample) is anchored or crosslinked to the swellable material before expansion. This can preferably be accomplished by chemically crosslinking a tag or label with the swellable material, such as during or after the polymerization or in situ formation of the swellable material. ”
With regards to claim 185, Chen teaches epifluorescence (0052).
Response to Arguments
The response traverses the rejection in view of the amendment. This argument has been thoroughly reviewed but is not considered persuasive as Trafton, Thomsen, Alon render the limitations obvious.
Claim(s) 179-181 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen (WO2015127183), Trafton (MIT news 1/15/2015) Thomsen (RNA (2005) volume 11, pages 1745-1748), Alon (2015 SCSB FALL POSTER SESSION ABSTRACTS)
as applied to claims 170-171, 173, 175-176, 178, 182-185 above, and further in view of Schiboleth (US20150211023).
The teachings of Chen, Trafton, Thomsen and Alon are set forth above.
Chen, Trafton, Thomsen and Alon do not specifically teach the acrydite modification of the 3’ or 5’ end.
However, Shiboleth teaches methods of modify nucleic acid sequences (abstract). Shiboleth suggests in paragraph 0036 that nucleic acids can be modified at the 5’ end, the 3’ end or at an internal location. Shiboleth continues in paragraph 0034 to identify modifications including acrydite.
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to substitute 3’ end acrydite modified nucleic acids or internally acrydite modified nucleic acids in the method of Chen, Trafton, Thomsen and Alon. The artisan would be motivated to determine if the location of the acrydite and or/ the level of expansion altered the specificity, sensitivity or location the target in the image following polymerization. The artisan would have a reasonable expectation of success the artisan is merely using art accepted and known reagents, conditions, and methods.
Response to Arguments
The response traverses the rejection in view of the amendment. This argument has been thoroughly reviewed but is not considered persuasive as Trafton, Thomsen, Alon render the limitations obvious.
Claim(s) 189 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen (WO2015127183), Trafton (MIT news 1/15/2015) Thomsen (RNA (2005) volume 11, pages 1745-1748), Alon (2015 SCSB FALL POSTER SESSION ABSTRACTS) as applied to claims 170-171, 173, 175-176, 178, 182-185 above, and further in view of Goldenberger (PCR Methods and Applications (1995) volume 4, pages 368-370)).
The teachings of Chen, Trafton, Thomsen and Alon are set forth above.
While Chen teaches, “Proteinase K (New England Biolabs) was diluted to 200ug/mL in digestion buffer (50mM Tris pH8, lmM EDTA, 0.5% TritonXlO0, IM NaCl, 0.8M guanidine HCl)” (0042)
Chen does not specifically teach the use of proteinase K and SDS.
However, Goldenberger teaches the use of SDS and proteinase K to digest proteins in a sample.
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to substitute SDS and proteinase K for SDS and Triton X100. The artisan would be motivated to use SDS with proteinase K as it is known to be an activator of proteinase K and disrupts membranes. The artisan would have a reasonable expectation of success as the artisan is merely substituting one detergent for another.
Response to Arguments
The response traverses the rejection in view of the amendment. This argument has been thoroughly reviewed but is not considered persuasive as Trafton, Thomsen, Alon render the limitations obvious.
Summary
No claims are allowed.
Conclusion
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 STEVEN C POHNERT PhD whose telephone number is (571)272-3803. The examiner can normally be reached Monday- Friday about 6:00 AM-5:00 PM, every second Friday off.
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/Steven Pohnert/ Primary Examiner, Art Unit 1683
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