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 .
Priority
This application is a CON of 18/188,504 (03/23/2023),
18/188,504 is a CON of 17/122,321 (12/15/2020, US11634751),
17/122,321 is a CON of 14/941,433 (11/13/2015, US10900065),
14/941,433 has PRO 62/080,055 (11/14/2014).
Claim Rejections - 35 USC § 103
Claims 1-6, 10-12, 14, 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over Ke et al. (Nat Methods 10, 2013-07-14, p. 857–860 and Supplemental, 35 pages) in view of Saliba et al. (Nucleic Acids Research, 2014-07-22, Vol. 42, No. 14, p. 8845–8860).
Ke teaches in situ sequencing in cells as depicted in Fig. 1a:
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through providing fixed and permeabilized cells (p. 5: “Fixation was performed in 3% (w/v) paraformaldehyde”, “The tissue was permeabilized in 2 mg/ml pepsin”), generating cDNA via RT using primers (p. 5: “In situ reverse transcription”, “cDNA primer or 5 μM of unmodified random decamers (all oligonucleotides sequences are listed in Supplementary Tables 4–6)”), tagging with barcode sequence (p. 5: “Barcode padlock probing”).
Regarding claim 1, Ke does not teach lysing the cells and isolation.
Saliba reviews single-cell RNA-seq (Title, Abstract) including utilizing the cell as a compartment to perform RNA-seq (p. 8851: “Sub-single-cell sequencing: localizing transcripts within a cell”, “A first strategy consists of isolating a compartment of a cell and applying the previously described RNA-seq techniques”). Saliba also describes details of the RNA-seq technique in Fig. 3 (p. 8851-52: “massively parallel RNA-seq of single cells (Figure 3)”) which when not performed within a cell includes lysis and isolation (Fig. 3: “Cell lysis” “bead immobilization”).
One of ordinary skill in the art following the teaching of Ke would have considered the teaching of Saliba which cites to Ke which are in the same field of endeavor of single cell sequence analysis. One of ordinary skill in the art would have considered combining the known technique of RNA-seq adapted for an in situ manner as taught by Ke and modify the steps to perform lysis and isolation after RT and tagging to maintain the integrity of the cell for use as a compartment as specifically suggested by Saliba. The level of skill in the art is very high as evidenced by the cited references and one of ordinary skill in the art routinely modifies and adapts known techniques to improve the information that can be derived from samples.
Regarding claims 2 and 3 further amplifying the isolated cDNA and sequencing, Ke teaches amplification and sequencing (p. 1, Fig. 1) as does Saliba (p. 8851, Fig. 3: “library preparation” and “library sequencing”) and one of ordinary skill in the art would consider applying the same steps as to obtain sequence information from the cell in the same manner as in the prior art.
Regarding claim 4 specifying RT primers comprise poly-T or a random sequence, Ke teaches random sequence (p. 3: “We generated cDNA from total RNA in situ by random-decamer priming”) and Saliba teaches oligo(dT) (Fig. 3) which corresponds to the claimed invention.
Regarding claim 5 specifying RT primers comprise a gene-specific sequence, Ke teaches gene-specific sequences (p. 27: Supp Table 4) as does Saliba (p. 8851: “Transcripts
are converted into cDNA using gene-specific (126) or random primers”) which corresponds to the claimed invention.
Regarding claim 6 to cell-specific tagging, Saliba teaches “Cellular and molecular barcoding strategies” (p. 8851) which localizes transcripts within a cell to facilitate cell-specific barcoding (p. 8851-52, Fig. 3) which corresponds to the claimed invention.
Regarding claims 10-11 specifying a biotin capture agent attached, Saliba specifically teaches such a configuration (Fig. 3A: “Streptavidin beads – Biot-…”) and one of ordinary skill in the art would have considered using the same configuration.
Regarding claim 12 specifying next generation sequencing, Saliba specifically teaches the use of NGS as applied to RNA-seq (p. 8845).
Regarding claim 14 specifying mammalian cells, Ke (p.1: “human breast cancer tissue”) and Saliba (p. 8846, 8856) both teach application of the technique on mammalian cells.
Regarding claims 18-20 specifying details regarding the cells, isolated, tissue, adherent, Saliba teaches isolated cells and Ke teaches adherent tissue cells (p. 1,18).
With each of the claims, the level of skill in the art is very high such that one of ordinary skill in the art would consider routine the combination of elements from the teaching of the art. One of ordinary skill in the art would have recognized that the results of the combination would be predictable due to the well-known nature and optimizations routinely performed in the art. Thus, one of ordinary skill in the art would have arrived at the invention as claimed before the effective filing date with a reasonable expectation of success.
Claims 7, 13 are rejected under 35 U.S.C. 103 as being unpatentable over Ke et al. (Nat Methods 10, 2013-07-14, p. 857–860 and Supplemental, 35 pages) in view of Saliba et al. (Nucleic Acids Research, 2014-07-22, Vol. 42, No. 14, p. 8845–8860) as applied to claims 1-6, 10-12, 14, 18-20 above and further in view of Nolan et al. (WO2012106385).
Regarding claim 7 specifying details regarding tagging comprising steps of dividing, coupling, and combining, Ke and Saliba do not teach the specific split-pool barcoding steps.
Nolan is in the same field of endeavor of single cell analysis with details regarding unique barcoding. Nolan teaches kits for tagging target molecules of nucleic acids from transcripts ([0069]: “kits for individually tagging cells”; [00250]-[00256]; claims 69-112; [00152]; [0063]; [00192]: “target nucleic acids may be obtained from a single cell.”; [00195]: “detect a novel transcript in order to diagnose or condition”). Nolan teaches fixing the cells ([0006]: “In some embodiments, the invention relates to methods for identifying whether a plurality of targets are in a plurality of cells comprising: binding to the targets a plurality of tags, wherein a tag comprises a code that represents a) the target identity and b) the identity of the cell in which tag is binding. ... In some embodiments, the cell is lysed or fixed.”). Nolan teaches permeabilizing the cells ([00116]: “Cells may be fixed prior to the addition of UBAs, ESBs or prior to COB assembly. Suitable cell permeabilization methods are known in the art and can be used to deliver components of the assay into cells and cellular components.”). Nolan teaches single-stranded tags / UBAs ([0010]: “In some embodiments, the tag comprises a UBA.”; [0082]: “In some embodiments, the UBA is an aptamer. Aptamers include nucleic acid aptamers (i.e., single-stranded DNA molecules …”) which are configured to hybridize to the transcriptome of the cell ([00229]: “the UBAs, e.g., oligonucleotide probe, have substantially the same length so that they hybridize to target nucleotide sequences at substantially similar hybridization conditions. As a result, the process of the present invention is able to detect infectious diseases, genetic diseases, and cancer”; [0064]; [00192]: “target nucleic acids may be obtained from a single cell.”; [00195]: “detect a novel transcript in order to diagnose or condition”) and has a second sequence that is the same in each of the nucleic acid molecules ([00110]: “The COB can be attached to the UBA via a common linker (CL). The CL can also be part of an oligonucleotide”). Nolan teaches a set of tags comprising multiple distinct barcode sequences comprising a sequence complementary to a common sequence ([00110]: “Substantially complementary or exact complementary annealing regions may be utilized for hybridization. An annealing region may be provided on both ends of an oligonucleotide ESB or APS. In some embodiments, the APSs are added in various steps of a split pool synthesis or any other suitable stepwise synthesis known in the art. An annealing region specific to each step of a stepwise synthesis maybe incorporated into the oligonucleotides”; [00111]-[00113]: “APSs can be designed to hybridize to the CL”). Nolan teaches ligation ([0021]: “the method further comprises ligation”; [0084]; [00121]; [00170]). Nolan teaches lysing the cell ([0006]: “In some embodiments, the cell is lysed”). Nolan teaches PCR amplification of DNA using polymerase ([0063]: “PCR amplification”; [00115]; [00125]: “the assembled products are amplified”, “products are amplified by polymerase chain reaction (PCR).”; [00191]; [00273]: “The COBs are optionally PCR amplified for sequencing using primers targeting the amplification primer complementary regions on the CL and the last APS subunit.”; [00170]: “extended via polymerases”; [0059]).
Nolan teaches the tags are combined in a manner to uniquely identify the target molecules with a “split pool” approach where the number of unique tags are generated by dividing, coupling an aliquot-specific barcode, and pooling (Fig. 4; [00163]-[00170]). One of ordinary skill in the art would have had a reasonable expectation of success in utilizing Nolan’s barcoding technique because Nolan teaches it in the same context (cellular barcoding) and it was well-known in the art.
Regarding claim 13 specifying ligation of tags, Saliba teaches the use ligation as applied to RNA-seq (p. 8845) as does Nolan ([0021]: “the method further comprises ligation”; [0084]; [00121]; [00170]) which one of ordinary skill in the art would consider routine in the art.
Claims 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Ke et al. (Nat Methods 10, 2013-07-14, p. 857–860 and Supplemental, 35 pages) in view of Saliba et al. (Nucleic Acids Research, 2014-07-22, Vol. 42, No. 14, p. 8845–8860) as applied to claims 1-6, 10-12, 14, 18-20 above and further in view of Koh (Chapter 13 - Isolation of Genomic DNA from Mammalian Cells, in Methods in Enzymology, Academic Press, Editor(s): Jon Lorsch, Volume 529, 2013, Pages 161-169).
Regarding claims 8-9 specifying the lysing agent comprises proteinase K, Ke and Saliba do not teach the lysis agent. Koh is a text on techniques in the art and states “isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique” and provides the lysis agent comprises proteinase K (p. 162). One of ordinary skill in the art would have considered using well-known and routine lysing agents as taught by Koh and arrived at the claimed invention with a reasonable expectation of success.
Claims 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Ke et al. (Nat Methods 10, 2013-07-14, p. 857–860 and Supplemental, 35 pages) in view of Saliba et al. (Nucleic Acids Research, 2014-07-22, Vol. 42, No. 14, p. 8845–8860) as applied to claims 1-6, 10-12, 14, 18-20 above and further in view of Islam (“From Single-Cell Transcriptomics to Single-Molecule Counting”, Thesis - Karolinska Institutet (Sweden). 2013. 59 pages).
Regarding claims 15-17, Ke and Saliba do not teach the particular primers, however, Islam teaches that the use of these elements are part of standard sequencing library preparation in in reverse transcription (p. 15: “In standard library preparation for RNA-seq experiments, the RT reaction is primed by different types of primer and different RNA priming strategy can effect the efficiency of cDNA synthesis. Three priming strategies are available: sequence-specific primer, oligo (dT) primer and random hexamer.”; p. 7: “primer designed with an anchored poly T”) such that one of ordinary skill in the art would have reasonably considered their use, optimized them for priming in the combined technique, and arrive at the claimed invention.
Response to Remarks - 35 USC § 103
Applicant argues that one of ordinary skill in the art would not be motivated to modify the method disclosed by Ke to incorporate lysis or cDNA isolation as taught by Saliba, as doing so would have negated the entire purpose of Ke' s approach. This argument is not persuasive because one of ordinary skill in the art following the teaching of Ke and Salida would recognize that in-cell sequencing was possible as demonstrated by Ke and would apply other known sequencing techniques for the same purpose, including RNA-seq as Salida teaches was successfully done for nuclei (“isolating a compartment of a cell and applying the previously described RNA-seq techniques”), in a manner that would be allow for in-cell sequencing.
Applicant argues the methods described by Ke and Saliba are in fact incompatible and cannot be combined without fundamentally altering their operation. This argument is not persuasive as both techniques are for sequencing cellular RNA and combining them would be within the technical grasp of one of ordinary skill in the art given the high level of skill as evidenced by Ke and Saliba. Combining two known strategies for the same purpose to obtain the same information would have been obvious to one of ordinary skill in the art.
Applicant argues that Salida was mischaracterized and does not teach or suggest in-cell RNA-seq and does not suggest using the cell as a compartment. This argument is not persuasive because Salida teaches under the heading “Sub-single-cell sequencing: localizing transcripts within a cell” (p. 8851):
… it would be insightful to sequence cellular transcripts while preserving their natural context.
A first strategy consists of isolating a compartment of a cell and applying the previously described RNA-seq techniques. This has been achieved for single nuclei (124) as well as dendrites from neurons (125). Another strategy consists of sequencing RNA directly inside a cell without lysis (126,127), a method called ‘in situ sequencing’.
Thus, Salida in citing Ke (ref 126) suggests performing RNA-seq in the context of “localizing transcripts within a cell” as well as “directly inside a cell without lysis” using known strategies - isolating a cell compartment and applying RNA-seq as well as sequencing directly inside a cell without lysis. One of ordinary skill in the art would have considered combining aspects of the strategies for the same purpose to maintain localization of the transcripts and arrive at the claimed invention with a reasonable expectation of success.
Applicant argues modifying Ke by introducing a lysis and cDNA isolation step would have removed the contextual information provided by sequencing the transcripts in situ and negated the purpose of the combination. This argument is not persuasive because one of ordinary skill in the art would have recognized that using RNA-seq in this manner would have maintained localizing transcripts within while they were tagged/barcoded to identify the cell’s transcripts/sequencing and preserve the purpose of the technique.
Applicant argues that Ke teaches away from the claimed invention because of Ke’s description of limitations of existing techniques that operate on homogenized samples that do not preserve localization of transcripts within a cell. This is not persuasive because it does not discourage localization and was actually providing a motivation for maintaining localization in a cell as also discussed by Salida.
Applicant argues the proposed modification would change the principle of operation of Ke from an in situ spatial sequencing method to an extraction-based sequencing workflow. This argument is not persuasive because one of ordinary skill in the art following the teaching of Ke and Salida would recognize that maintaining localization of transcripts within a cell is beneficial and in view of the success performing RNA-seq in nuclei would have had a reasonable expectation of success in applying the same technique to a cell.
None of Applicant’s arguments are persuasive as to the nonobviousness of the claims and the rejections are maintained.
Double Patenting
Applicant filed an approved terminal disclaimer; thus, the double patenting rejections are withdrawn.
Conclusion
No claims allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/ROBERT H HAVLIN/Primary Patent Examiner, Art Unit 1626
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