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 .
Election/Restrictions
Applicant's election with traverse of group I, in the reply filed on 6/22/2026is acknowledged. The traversal is on the ground(s) that the response traverses the rejection asserting the restriction asserting the statements in the restriction asserting the methods have different steps and limitations and are thus distinct. This is not found persuasive because the response has merely asserted the distinction analysis is conclusory, but has not demonstrated that the different steps and limitations does not make the inventions distinct . Thust the restriction is maintained.
The response further traverses the species election of poly A asserting, “Poly A, random sequences, and universal bases are not patentably distinct species and each are capable of hybridizing the immobilization oligonucleotide to the target nucleic acid with the same base-pairing principles.” This argument has been thoroughly reviewed but is not considered persuasive as Poly A, random sequences, and universal bases have different nucleotides and searching one will not provide art on the other. If applicant wishes to on the record indicate each one is patentably indistinct the species election, may be withdrawn.
The response traverses the election of Thymine. The response traverses the rejection asserting, “ The four different bases are not patentably distinct species and each are capable of base-pairing and serve the same function for hybridization.” This argument has been thoroughly reviewed but is not considered persuasive as A, , C T and G are different nucleotides and searching one will not provide art on the other. . If applicant wishes to on the record indicate each one is patentably indistinct the species election, may be withdrawn.
The response traverses the election of 3-cyanovinylcarbazole (CNVK) nucleoside. The response traverses the rejection asserting, “ he enumerated crosslinkable moieties (3-cyanovinylcarbazole (CNVK) nucleoside, 3-cyanovinylcarbazole modified D-threoninol (CNVD), pyranocarbazole nucleoside (PCX), pyranocarbazole modified D-threoninol (PCXD), psoralen and its derivatives) are all photo-activated crosslinking agents that operate by similar mechanism of covalent.” This argument has been thoroughly reviewed but is not considered persuasive searching one of the crosslinkers will not inherently provide art on the others. . If applicant wishes to on the record indicate each one is patentably indistinct the species election, may be withdrawn.
The applicant further elects” Applicant elects: (4) an immobilization oligonucleotide comprising three nucleotide residues functionalized with crosslinkable moieties in the hybridization region; (5) an attachment moiety that is an acrydite; (6) the attachment moiety being attached to an anchoring moiety in the matrix; (7) the attachment moiety and the anchoring moiety being functional moieties that can react with each other; (8) the primary probe or probe set being a circularizable probe or probe set; (9) crosslinking the one or more modified crosslinkable nucleotide residues in the RCP to the biological sample or the matrix; (10) detecting in (d) signals associated with detectably labeled probes that are hybridized to barcode regions or complements thereof in the primary probe or probe set or a product thereof; (11) amplifying the signal by rolling circle amplification (RCA) of a probe that directly or indirectly binds to the primary probe or probe set and/or the amplification product thereof; (12) the matrix being functionalized with the anchoring moiety to bind covalently to the attachment moiety; (13) an anchoring moiety reactive group that is acrydite; (14) a second attachment moiety that is photo-crosslinkable; and (15) a biological sample that is a formalin-fixed, paraffin-embedded (FFPE) sample. These elections are made without traverse.”
Claims 10-12, 22, 26-30, 33-37, 41, 54, 60, 66, 80, 114, 126, 130 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected inventions or species, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 4/28/2025.
Claims 1-9, 13-21, 23-25, 31, 32, 35, 38-40, 42-53, 55-59, 61-65, 119, and 136-146 are being examined.
Priority
The instant application was filed 03/14/2024 and claims priority from provisional application 63502295 filed 05/15/2023 which claims priority from provisional application 63496887 filed 04/18/2023; claims priority from provisional application 63491516 filed 03/21/2023; and claims priority from provisional application 63490870 , filed 03/17/2023.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 5/5/2025 is being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered..
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) 1-9, 13-15, 19, 38-40, 56-58, 136-146 is/are rejected under 35 U.S.C. 103 as being unpatentable over Deisseroth (WO 2017/139501) and Tentori (US 2021/0189475)
MPEP 2111.01 II states:
The problem is to interpret claims ‘in view of the specification’ without unnecessarily importing limitations from the specification into the claims."); Altiris Inc. v. Symantec Corp., 318 F.3d 1363, 1371, 65 USPQ2d 1865, 1869-70 (Fed. Cir. 2003) (Although the specification discussed only a single embodiment, the court held that it was improper to read a specific order of steps into method claims where, as a matter of logic or grammar, the language of the method claims did not impose a specific order on the performance of the method steps, and the specification did not directly or implicitly require a particular order).
Thus the broadest reasonable interpretation of the claims is the steps can be done in any order.
With regards to claim 1, Deisseroth teaches, “An aspect of the present disclosure includes a method of preparing a biological specimen for microscopic analysis of a target RNA analyte, the method including fixing the specimen with a plurality ofhydrogel subunits; polyrnerizing the hydrogel subunits to form a hydrogel-embedded specimen; fixing RNA in the specimen using ca.rbodiimide mediated crosslinking; clearing the hydrogel-embedded specimen wherein the RNA is substantially retained in the specimen; and contacting the specimen with a nucleic acid probe for a target RNA analyte.” (0007)
The courts have haled that rearrangement of steps is obvious in the absence of unexpected results. Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also 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); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.).
Deisseroth does not specifically teach an attachment moiety.
However, Tertori teaches use of acrydite modified oligonucleotides to attach anchor the oligonucleotides to gels. (0678-0679, )
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 modify the method of Deissorth by adding an acrydite moiety to the immobilization oligonucleotide and providing the oligonucleotide prior to polymerization. The artisan would be motivated to add the acrydite moiety to the oligonucleotide and provide the oligonucleotide prior to polymerization to anchor the target in location prior to polymerization to allow for determination of spatial location. The artisan would have a reasonable expectation of success as the artisan is merely using known oligonucleotide modifications to anchor the oligonucleotide and its target in place.
With regards to claim 2, Deisseroth teaches, “the nucleic acid probe undergoes a Hybridization Chain Reaction (HCR)….. In some embodiments, the contacting comprises contacting the specimen with a plurality of nucleic acid probes for a plurality of target RNA analytes.” )0008)
With regards to claim 3, Deisseroth teaches detection of the RNA (0051).
With regards to claim 4-8,13 Deisseroth teaches, “we performed in situ hybridization with a 50 base deoxy-thymine oligonucleotide (oligo(dT)) to target the poly A tail of mature mRNA “ (00107)
With regards to claim 9, Tertori teaches Fragmented RNA or DNA (0388)
With regards to claim 14, Deisseroth teaches probes can be PNA which are not extendable by a polymerase (0049)
With regards to claim 15, Tertori teaches 3’ modification of oligonucleotides include dideoxy modification.)(0325)
With regards to claim 19, Deisseroth teaches photocrosslinking (0033)
With regards to claim 38- 40, Tertori teaches use of acrydite modified oligonucleotides to attach anchor the oligonucleotides to gels. (0678-0679
With regards to claim 56, Deisseroth teaches acrylamide 0080
With regards to claim 57,58 the attachment of the acrydite makes a covalent attachment to the matrix.
With regards to claim 136-138, Deisseroth teaches PFA matrix formation followed by hydrogel formation. (0081)
With regards to claim 139-140, Deisseroth teaches acrylamide hydrogel (0080)
With regards to claim 141, Deisseroth teaches formalin (0105)
With regards to claim 142, 144-145, Deisseroth teaches, “Once the RNA is fixed, the hydro-gel embedded specimen may be cleared. By "clearing" a specimen it is meant that the specimen is made substantially transparent i.e., permeable to light.” (0043)
With regards to claim 143, Deisseroth teaches PFA matrix formation prior to hydrogel formation.
With regards to claim 146, Deisseroth teaches, “In certain cases, the tissue specimen may be at least about 0.1 mm thick, such as, about 10 mm-0.lmm, 8 mm-1 mm, 6 mm-lmm, 4 nun-lmm, 3 mm-lmm, 3 mm-0.Smm, 3 mrn-0.3mm, 3 mrn-0. l mm, 5 mm-0.5mm, 5 mm-0.3mm, 5 mm-0. l mm, l O mm- 0.3mm, or l O mm-0 1 mm thick.” (0068)
Claim(s) 16-21, 23-25, 31-32, 35 are rejected under 35 U.S.C. 103 as being unpatentable over over Deisseroth (WO 2017/139501) and Tentori (US 2021/0189475 as applied to claims 1-9, 13-15, 19, 38-40, 56-58, 136-146 above, and further in view of Mochizuki ( Journal of Biotechnology (2015) volume 212, pages 174-180) and Nemoto (US20180100146).
The teachings of Deisseroth and Tentori are set forth above.
While Deisseroth and Tentori suggest the methods of the claims. They do not specifically teach the crosslinking agent is on the nucleotide or CNVK.
However, Deisseroth teaches photo-crosslinking.
Mochizuki teaches a method of cDNA display. Mochizuki teaches, “we introduce a novel Pu-linker containing 3 cyanovinylcarbazole nucleoside (cnvK) named the cnvK-Pu-linker. cnvK is an ultrafast photo-cross-linker which connects hybridized oligonucleotides using 366 nm of UV irradiation (Yoshimura and Fujimoto, 2008; Yoshimura et al., 2009). This method enables ultra-fast cross-linking of the developed Pu-linker to the 3′-terminal region of an mRNA without the use of an enzymatic ligation reac tion.” (page 175, 1st column, middle). Mochizuki teaches, “he cnvK-Pu-linker can be photo-cross-linked with mRNA using 30 s of UV irradiation (Fig. 2A), making it the fastest fusion of a Pu-linker to an mRNA to the best of our knowledge. Addi tionally, degradation of the mRNA was suppressed compared with the conventional SBS-Pu-linker as shown in Fig. 3. Smeared bands were detected under the band of linker-ligated mRNA (562 mer) when SBS-linker was used, which could be the result of mRNA degradation during ligation reaction. Conventional Pu-linkers can be ligated to the 3′-terminus of mRNA using T4 DNA or RNA lig ase in a ligation buffer containing 10 mM of MgCl2 (Olson et al., 2008; Yamaguchi et al., 2009; Mochizuki et al., 2011),which can also activate ribonucleases thus linker-ligated mRNAs can be eas ily degraded by ribonuclease contamination. Instead, cnvK can be photo-cross-linked under divalent cation-depleted conditions, which is intrinsically advantageous for handling mRNA. We also confirmed that omitting cross-linking by UV irradiation critically decreases the fusion efficiency of the mRNA with the nascent pro tein as shown in Fig. 2B. This may be attributed to breaking of the hybridization between the mRNA and cnvK-Pu-linker by ribosome helicase activity as previously mentioned by other groups “
Nemoto teaches, “ the high-speed photo-cross-linking site being composed of cnvK makes possible to conduct photo-cross-linking reaction without any enzymes in water, wherein the enzyme cannot function. Therefore, the decomposition of mRNA by RNase is prevented.”(0083)
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 CNVK of Mochizuk and Nemoto i in the method of Deisseroth and Tentori. The artisan would be motivated to use CNVK as the crosslinker as Mochizuki teaches it is ultra-fact and under divalent cation depleted conditions. The artisan would have a reasonable expectation of success as the artisan is substituting one crosslinker for one with better properties.
With regards to claims 16-18, 23-25, 35, CNVK crosslinks to a nucleotide based of hybridized sequence (figure 1).
With regards to claims 20, Mochizuki teaches CNVK crosslinks at 366 nm of UV irradiation (175, 1st column, 2nd full paragraph).
With regards to claim 21, CNVK crosslinks pyrimidines.
While Mochizuk and Nemoto suggest the use of CNVK, they do not specifically teach more than one CNVK for immobilization probe. However, 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 provide multiple CNVK in a single immobilization probe or ofigonucleotide. Thea artisan would be motivated to provide multiple CNVK to allow for multiple sites of attachment between the target sequences and the immobilization probe. Thea artisan would have a reasonable expectation of success as the artisan is merely providing multiple CNVK in a single probe.
Claim(s) 42-53 55, 59, 61-65, 119 are rejected under 35 U.S.C. 103 as being unpatentable over Deisseroth (WO 2017/139501) and Tentori (US 2021/0189475 as applied to claims 1-9, 13-15, 19, 38-40, 56-58, 136-146 above, and further in view of Gao (Trends in Analytical Chemistry Volume 121, December 2019, 115700), Chen (US 20190360044), Church (US 20170159136), Glen Report Research (Glenreseearch.com (2019).
The teachings of Deisseroth and Tentori are set forth above.
While Deisseroth and Tentori suggest the methods of the independent claims. They do not specifically teach the circularizable probes or rolling circle amplificaiton.
However, Chen teaches, “[0042] In some embodiments, the proximity dependent probes are molecular inversion probes (MIPs), HyPR probes, padlock probes, or split-ligation probes, each probe further comprising a unique molecular identifier (UMI).” Chen teaches, “0136] FIG. 5—In situ MIP readout via rolling circle amplification and subsequent fluorescent sequencing. Each set of MIPs for a given RNA transcript is given a unique sequence barcode which can be read out via sequencing by ligation (SOLiD), this enables 4′N multiplexing of transcripts where N is the number of sequencing bases (3 bases shown here). Bottom, allele specific in situ detection of SNP variants via MIPs.”
Gao teaches provides a review of rolling circle amplification. Gao teaches in situ RCA (figure 2).
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 MIPs or padlock probes for the probes of Deisseroth and Tentori. The artisan would be motivated as Gao teaches, “irements for expensive microscopy setups. In this review, we highlight the RCA-based approach for highly efficient, specific and multiplex amplification of fluorescent signals in situ in single cells.” The artisan would have a reasonable expectation of success as the artisan is substituting one probe and detection method for another.
With regards to claims 42-44, Goa and Chen teach circularizable probes and generation of rolling circle amplification products(throughout).
The specification does not teach a definition of orthogonal to attachment moiety. Thust eh broadest reasonable interpretation is attached.
Chen teaches, “We covalently couple nucleic acids and proteins to the polyacrylamide hydrogel using various small molecule anchoring approaches we have previously developed. This facilitates easy reagent and buffer changes and allows molecular biology steps to be performed in a standard Eppendorf test tube on millions of cells in parallel, while retaining each cell's contents in a compartmentalized hydrogel droplet.” (0523)
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 attach the primer to the biological sample or orthogonal to the attachment moiety. Thea artisan would be motivated as Chen suggest anchoring the nucleic acids and proteins to hydrogels. The artisan would have a reasonable expectation of success as Chen teaches they have previously developed such reagents.
Deisseroth, Tentori, Gao and Chen do not specifically teach crosslinkable nucleotides in RCP products.
However, Church teaches, “In certain aspects, the DNA is circularized using CircLigase and amplified using Phi29 DNA polymerase. Crosslinker compatible nucleotides are incorporated during reverse transcription and rolling circle amplification. Crosslinkers can then be used to attach nucleotides to a subcellular component (e.g., the cell matrix and/or one or more proteins and/or attached to a synthetic three-dimensional support matrix (e.g., co-polymerized in an acrylamide gel). The single molecule amplicons are sequenced using direct DNA ligation, extension, or hybridization using fluorescently labelled probes. The sequential images from multiple sequencing or hybridization cycles are used to generate sequencing reads from each protein-RNA complex. The barcode sequence is then used to identify individual proteins and where the RNA is transcribed.”
Glen Report teaches BRDU as a photocrosslinker.
Therefore it would have been prima facie obvious to one of skill in the art prior to the effective filing date of the claims to use crosslinkable nucleotides analogs including BrdU in the RCP methods of Deisseroth, Tentori, Gao and Chen. Thea artisan would be motivated to have the RCP products be attached to a matrix. Thea artisan would have a reasonable expectation of success as the artisan is merely using known nucleotide analogs in known methods. (47-48, 59, 61-65)
With regards to claim 49-53, 55 the art of Gao, Chen and Church demonstrates methods of detecting RCP products by direct or indirect labeling with intermediate probes using fluorescence was known.
Summary
No claims are allowed.
Conclusion
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/Steven Pohnert/ Primary Examiner, Art Unit 1683
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