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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 12/20/2023, 02/14/2024, 04/03/2024, 04/16/204, 06/20/2024, 07/15/2024, 08/02/2024, 09/10/2024, 09/27/2024, 10/14/2024, 11/26/2024, 12/06/2024, 05/22/2025, 07/30/2025, 04/24, 2026 are acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Status
Claims 1-30 are pending. Claims 1-30 are under examination and discussed in this Office Action.
Specification
The instant specification does not include an Example 17. The instant specification proceeds directly from Example 16 [00207] to Example 18 [00208], skipping Example 17 entirely.
Claim Objections
Claim 9 is objected to because of the following informalities:
Claim 9 recites “wherein the ratio of SPRI bead solution to amplified cDNA solution is between about 0.9:1 and about 0:7:1.” “0:7:1” should instead be “0.7:1” consistent with the instant specification at paragraph [0082] and [0097].
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 1-30 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 1 (f)ii) recites “a 3’ hybridization sequence located 3’ of the barcode sequence and/or a 5’ hybridization sequence located 5’ of the barcode sequence,” The use of “and/or” renders the claim indefinite because it encompasses three distinct structural alternatives: a nucleic acid tag comprising only a 3’ hybridization sequence, a nucleic acid tag comprising only a 5’ hybridization sequence, and a nucleic acid tag comprising both without making clear which alternative or alternatives are required. The three alternatives define structurally distinct tag method that would function differently in the ligation mechanism described in the specification, and the claim as written does not establish which method or methods are encompassed.
Claim 2 recites “isolating the tagged cDNA molecules released during step (h) using a binding agent, such that the isolated tagged cDNA molecules are bound to the binding agent.” However, step (h) of claim 1 is a dividing step, not a lysis step. Step (h) recites “dividing the combined cells from the second plurality of aliquots into a plurality of samples, wherein each sample of the plurality of samples comprises more than one cell” No cDNA molecules are released during a dividing step. The tagged cDNA molecules are released by lysis, which occurs in step (i): “lysing the cells in the plurality of samples, thereby releasing the tagged cDNA molecules and forming a lysate in each sample of the plurality of samples.” Claim 2 therefore incorrectly cross-references step (h) as the source of the released tagged cDNA molecules when the release occurs in step (i).
Claim 28 recites the limitation "the nucleic acid tags used in the plurality of aliquots comprise 96 distinct barcode sequences" in lines 15-16. The phrase “the plurality of aliquots” lacks clear antecedent basis in claim 28. Claim 1 from which it depends introduces two distinct pluralities of aliquots: “a first plurality of aliquots” in step (b) and “second plurality of aliquots” in step (e). the reference to “the plurality of aliquots” in claim 28 is ambiguous as to whether it refers to the first plurality of aliquots, the second plurality of aliquots, or both. Because the RT primer barcode sequences are specific to aliquots of the first plurality and the nucleic acid tag barcode sequences are specific to aliquots of the second plurality, this ambiguity is material to the scope of the claim. There is insufficient antecedent basis for this limitation in the claim.
Claims 3-7, 9-25, and 28-30 are also rejected for their dependence on claims 1, 2, 8, 27, or 28 and do not further clarify the indefiniteness identified above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. 11,680,283 (16/649,601). Although the claims at issue are not identical, they are not patentably distinct from each other.
The claims of ‘283 patent is directed to a method of uniquely labeling RNA molecules within a plurality of cells comprising fixing and permeabilizing cells at below about 8oC, reverse transcribing RNA with RT primers comprising a poly(T) or random sequence, split-pool rounds with primary and secondary nucleic acid tags, lysis, and adding a protease inhibitor and binding agent to the lysate. Dependent claim 7 of ‘283 patent further requires that the RT primers comprise a 5’ overhang and that each nucleic acid tag comprises a first strand with a barcode sequence flanked by a 3’ hybridization sequence and a 5’ hybridization sequence and a second strand comprising portions complementary to the 5’ overhang sequence and the 3’ hybridization sequence.
The instant claim 1 recites these same elements: 8oC fixation, poly(T) or random RT primers, split-pool barcoding, lysis, and additionally requires in the independent claim the 5’ overhang RT primer architecture and the nucleic acid tag hybridization sequence structure that appear in dependent claim 7 of ‘283 application. The combination of claims 1 and 7 of ‘283 patent covers the same method as instant claim 1.
Instant claim 1 differs from the combination of ‘283 claims 1 and 7 in that the instant claim 1 does not require a protease inhibitor and binding agent in the independent claim, and explicitly requires aliquot-specific RT primer barcode sequences and the dividing of combined cells into a plurality of samples before lysis. These differences represent obvious variation, omitting the protease inhibitor from the independent claim while retaining it in a dependent claim (instant claim 4) does not create a patentable distinction, and adding the explicit dividing step and aliquot-specific barcode language merely recites steps that are inherent in obviously implied by the split-pool method already claimed in ‘283 patent. Incorporating features of a dependent claim into the independent claim of a continuation to create a new independent claim constitutes an obvious variation of the already patented invention that does not give rise to a separate right to exclude. The instant claims 1-30, taken as a whole, define a method that is an obvious species of the broader method already claimed in ‘283 patent.
Claims 1-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-27 of U.S. Patent No. 12,252,730 (18/304,670). Although the claims at issue are not identical, they are not patentably distinct from each other.
The claims of ‘730 patent are directed to a method of cell-specifically labeling RNA molecules within cells comprising fixing and permeabilizing cells at below about 8oC, split-pool barcoding rounds with RT primer barcodes specific to each aliquot and nucleic acid tag barcodes specific to each aliquot, lysis, and adding a protease inhibitor and binding agent to the lysate. Dependent claims 12 and 13 of ‘730 patent further require that the nucleic acid tags are brought into proximity of the 5’ end of the cDNA by prehybridization with a linker nucleic acid strand, and that the RT primers each comprise a 5’ overhang sequence that is present at the 5’ end of each cDNA molecule following reverse transcription.
Instant claim 1 recites the same split-pool cell-specific RNA labeling method and incorporates into the independent claim the 5’ overhang RT primer and hybridization-facilitated ligation tag structure that appear only in dependent claims 12-13 of ’730 patent. The combination of claims 1 and 12-13 of ‘730 patent covers the same method as instant claim 1.
The instant claim 1 differs from the combination of ‘730 claims 1 and 12-13 in that the instant claim 1 does not require a protease inhibitor and binding agent in the independent claim, and recites a dividing of the combined cells into a plurality of samples before lysis as part of the independent claim rather than as a dependent claim. These differences are obvious variations for the same reasons discussed above regarding ‘283 patent. Incorporating features of dependent claims into the independent claim of a continuation to create a new independent claim constitutes an obvious variation of the already patented invention that does not give rise to a separate right to exclude. The instant claims 1-30, taken as a while, define a method that is an obvious species of the method already claimed in ‘283 patent.
Claims 1-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 12,371,735 (19/053,925). Although the claims are not identical, they are not patentably distinct from each other.
The claims of ‘735 are directed to a method of preparing tagged cDNA molecules for use in single-cell transcriptome analysis comprising: fixing and permeabilizing cells at below about 8oC, dividing into a first plurality of aliquots each comprising more than one cell, reverse transcribing RNA molecules within the cells using RT primers each comprising a poly(T) or random nucleotide sequence, and RT primer barcode sequence specific to each individual aliquot, and a 5’overhabg sequence that is the same in all RT primers and is present at the 5’ end of each cDNA molecule following reverse transcription, combining the cells, dividing into a second plurality of aliquots, coupling nucleic acid tags comprising a tag barcode sequence specific to each individual aliquot and a 3’ hybridization sequence and/or a 5’ hybridization sequence to the cDNA molecules, combining the cells, lysing, and isolating the tagged cDNA molecules using a binding agent.
Comparing the claims of ‘735 to the instant claim 1, the two independent claims recite substantially the same method directed to cells with the same 8oC fixation, 5’ overhang RT primer architecture, and hybridization sequence nucleic acid tag structure. The primary structural difference is that instant claim 1 recites an explicit step (h) of dividing the combined cells into a plurality of samples before lysis, and step (i) of lysing the cells in the plurality of samples, which claim 1 of ‘735 does not recite the dividing steps in the independent claim. However, incorporating the feature of dependent claim 6 of ‘735 into the independent claim of the instant application constitutes an obvious variation that does not give rise to a separate right to exclude.
Additionally, instant claim 1 does not require isolating the tagged cDNA molecules using a binding agent in the independent claim, whereas ’735 patent claim 1 step (i) requires such isolation. This difference is also an obvious variation since isolation using a binding agent is recited in instant dependent claim 2. The instant claim 1-30, taken as a whole, define a method that is an obvious species of the method already claimed in ‘735 patent.
Claims 1-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-27 of U.S. Patent No. 12,252,731 (18/304,695) in view of Cao et al (2017).
The claims of ‘731 patent is directed to a method of nucleus-specifically labeling RNA molecules within nuclei comprising: fixing and permeabilizing a plurality of nuclei at below about 8oC, dividing the nuclei into a first plurality of aliquots each comprising more than one nucleus; reverse transcribing RNA molecules within the nuclei using RT primers comprising a poly(T) or random nucleotide sequence and an RT primer barcode sequence specific to each aliquot, pooling the nuclei, dividing into a second plurality of aliquots, coupling nucleic acid tags comprising a tag barcode sequence specific to each aliquot to the cDNA molecules, pooling, lysing, and adding a protease inhibitor, and a binding agent to the lysate. These steps are structurally identical to those recited in instant claims 1-30, with the sole distinction that ‘731 operates on nuclei while the instant claims operate on cells.
Although the instant claims are directed to cells and claims of ‘731 patent are directed to nuclei, this distinction does not provide a patentable distinction for purposes of nonstatutory double patenting. Cao explicitly discloses that “sci-RNA-seq is also compatible with nuclei, which may be important for tissues for which unbiased cell disaggregation protocols are not well established” and reports that gene expression measurements from aggregated transcriptomes of nuclei and cells were well correlated (Pearson r = 0.96 for HEK293T and 0.97 for NIH/3T3) (p. 7, column 1, para 3; Fig. 1E). It would have been obvious to one of ordinary skill in the art at the time the invention was made to apply the nucleus-specific labeling method of ‘731 patent to intact cells, or conversely to apply the cell-specific labeling method of the instant claims to nuclei, as the choice between intact cells and isolated nuclei was a routine experimental decision with predictable results well known in the single-cell RNA-seq field. The skilled artisan would have recognized cells and nuclei as known alternative substrates for performing the same split-pool combinatorial barcoding workflow, and the substitution of one for the other represents nothing more than the application of known techniques to a known variation with predictable results.
Claims 1-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 12,234,501 (18/398,901) in view of Cao et al (2017).
The claims of ‘501 patent are directed to a method of labeling RNA molecules with nucleus-specific tags comprising: fixing and permeabilizing a plurality of nuclei at below about 8oC, dividing into a first plurality of aliquots each comprising more than one nucleus, reverse transcribing RNA molecules within the nucleic using RT primers each comprising a poly(T) or random nucleotide sequence, an RT primer barcode sequence specific to each individual aliquot, and a 5’ overhang comprising a 5’ overhang sequence that is the same in all RT primers and is present at the 5’ end of each cDNA molecule following reverse transcription, combining, dividing into a second plurality of aliquots, coupling nucleic acid tags comprising a tag barcode sequence specific to each individual aliquot, and a 3’ hybridization sequence and/or a 5’ hybridization sequence to the cDNA molecules, combining, dividing into a plurality of samples each comprising more than one nucleus, and lysing the nuclei in the plurality of samples.
Comparing the claims of ‘501 patent to instant claim 1 element by element, the two independent claims are structurally identical in every respect. Every limitation of instant claim 1 including 8oC fixation, the 5’ overhang RT primer method with a common 5’ end of each cDNA molecule, the nucleic acid tags with a tag barcode sequence and flanking hybridization sequences, the dividing into a plurality of samples, and the lysis is present in claim 1 of ‘501. The sole distinction between the two independent claims is that ‘501 application claim 1 operates on nuclei while instant claim 1 operates on cells.
Cao establishes that applying combinatorial indexing single-cell RNA-seq methods to intact cells versus isolated nuclei was a routine choice with predictable results known to skilled artisans in the field (p. 7, column 1, para 3; Fig. 1E). A skilled artisan would have found it obvious to perform the nucleus-specific tagging method of ‘501 application on intact cells rather than nuclei, or conversely to perform the cell-specific tagging method of the instant claims on nuclei rather than intact cells, as cells and nuclei were recognized interchangeable substrates for split-pool combinatorial barcoding RNA-seq workflows.
The instant claims 1-30 and the claims of ‘501 therefore define obvious variants of the same inventions differing only in the choice of cellular substrate.
Claims 1-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-26 of U.S. Patent No. 12,227,793 (18/536,721). Although the claims are not identical, they are not patentably distinct from each other.
The claims of ‘793 patent are directed to a broad method of preparing nucleic acid library for single-cell transcriptome analysis comprising: fixing and permeabilizing a plurality of cells, reverse transcribing the transcriptome of each cell within the cells to generate cDNA molecules, coupling nucleic acid tags to the cDNA molecules within each cell to generate tagged cDNA molecules, lysing the cells and releasing the tagged cDNA molecules to form a lysate, isolating the released tagged cDNA molecules from the lysate, amplifying the isolated tagged cDNA molecules, and sequencing the amplified tagged cDNA molecules.
The instant claims 1-30 define a method that falls within the scope of claim 1 of ‘793 patent. Every step recited in instant claim 1: fixing and permeabilizing cells, reverse transcribing RNA to generate cDNA within the cells, coupling nucleic acid tags to the cDNA within the cells, lysing to release tagged cDNA, and isolating satisfies the corresponding steps of the ‘793 patent. The instant claims add specific limitations not expressly required by claim 1 of the ‘793 patent, including the 8oC fixation temperature, the split-pool aliquot-specific barcodes, the 5’ overhang RT primer architecture, and the hybridization sequence tag structure. However, these additional limitations represent obvious refinements of the general cell transcriptome labeling method of the ‘793 patent because: (i) the split-pool aliquot structure with aliquot-specific barcodes is expressly disclosed in dependent claims 6 and 7 of the ‘793; (ii) the use of protease for lysis and streptavidin bead isolation is disclosed in dependent claim 8-9 and 12-13; (iii) the amplification with sample-specific index sequences is disclosed in dependent claim 14; and (iv) the 8oC fixation temperature and 5’ overhang RT primer architecture are obvious optimizations of the general method that a skilled artisan would apply to improve RNA preservation and enable ligation-based barcode coupling. The instant claims 1-30, taken as a whole, define a method that is an obvious species of the broader method already claimed in ‘793 patent.
Prior Art
Claims 1-30 are free of prior art. The closes art Cao et al., Comprehensive single-cell transcriptional profiling of a multicellular organism. Science 357,661-667 (2017). DOI: 10.1126/science.aam8940, which discloses a combinatorial indexing method (sci-RNA-seq) for single-cell RNA sequencing comprising fixing and permeabilizing cells, distributing cells into wells, performing in situ reverse transcription using well-specified barcoded polythymidine primers, pooling and redistributing cells for a second round of indexing via Tn5 transposes tagmentation and indexed PCR, and lysing cells to generate sequencing libraries. However, Cao does not teach or suggest reverse transcription primers comprising a 5’ overhang sequence that is the same in all RT primers used in a given aliquot and that remains present at the 5’ end of each cDNA molecules following reverse transcription. Cao instead introduces the second barcode by Tn5 transposase tagmentation of double-stranded cDNA or by indexed PCR, rather than by hybridizing and ligation of a nucleic acid tag to a 5’ overhang on the cDNA. Cao also does not expressly disclose fixing and permeabilizing cells at a temperature below about 8oC. Therefore, Cao fails to anticipate or render obvious the claimed invention.
Conclusion
All claims are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Nura Choudhury whose telephone number is (571)272-6148. The examiner can normally be reached M-F, 9-5 ET.
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/NURA M. CHOUDHURY/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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