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 .
Status of the Application
Claim(s) 24-46 are pending and under examination
The following Office Action is in response to Applicant's communication dated 07/28/2026.
Any previous rejection(s) and/or objection(s) not repeated in this action are withdrawn. The rejection(s) and/or objection(s) maintained below, along with any newly applied one(s), constitute the complete set presently being applied to the instant application.
Response to Arguments
RE: Applicant argues the amended limitations distinguished the claimed subject matter from Song and thus, overcome the rejections of the amended claims and claims depending therefrom based on Song alone or in combination with Di and Zhu.
In response: Newly added limitations by Applicant in present amendment, which are considered and will be addressed in present office action.
RE: Applicant argues Di’s cell-free RNA-DNA hybrid tagmentation does not remedy Song’s deficiency because Di does not disclose that a transposase can selectively fragment an RNA-cDNA hybrid while avoiding genomic DNA.
In response: As presently drafted, claim 28 is much broader and not commensurate with the invention in Applicant’s argument. Applicants appear to rely on a specific transposase that exclusively fragment an RNA-cDNA hybrids and not dsDNA. However, as presently drafted, this limitation is not explicitly mentioned in the claim. 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, 26 USPQ2d 1057 (Fed. Cir. 1993).
Modified Claim Rejections - 35 USC § 103-Neccessitated by amendment
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Song et al. and Chen et al.
Claim(s) 24-27, 29-41, 43-45 is/are rejected under 35 U.S.C. 103 as being unpatentable over Song et al. (US11939622B2, EFD: July 22nd 2019, of record) in view of Chen et al. (WO2020186094A1, EFD: March 12th 2020).
Regarding claim 24, Song discloses a method for generating labeled nucleic acid molecules,
comprising the following steps: (a) providing multiple bead particles comprising coupled oligonucleotide molecules, wherein said oligonucleotide molecules contains a labeling sequence comprising a second label sequence, wherein each bead particle has multiple oligonucleotide molecules, and the multiple oligonucleotide molecules on the same bead particle have the same second label sequence, while oligonucleotide molecules on different bead particles have different second label sequences; (e.g. the bead is conjugated to a UMI [Fig 12] and plurality of oligonucleotide can be immobilized on the synthetic
particle (e.g. beads) [column 83, lines 19-20]. And "the cell label is identical for all barcodes attached to
a given solid support (e.g., a bead), but different for different solid supports (e.g., beads)." [column 31,
lines 28-30])
(b) providing multiple permeabilized cells or cell nuclei comprising RNA [claim 10];
(d) intracellular tagmentation, wherein the transposon sequence contains a transfer chain and a non-transfer chain, the transfer chain comprising a transposase recognition sequence, a first label sequence, and a first common sequence, wherein the first label sequence is located upstream of the transposase recognition sequence, and the first common sequence is located upstream of the first label sequence (e.g. contacting double-stranded nuclear target associated DNA with a conjugate comprising a transposome including a transposase and adaptor sequences to generate fragmented dsDNA having adaptor-derived 5' overhangs. The transposome includes a first adaptor and a second adaptor, each comprising barcode sequences. The adaptor sequence further includes sequencing adaptor and universal primer handle regions, corresponding to common sequences. Because adaptor-loaded Tn5 transposase inserts these adaptors transfer strands into fragment termini during tagmentation, Song discloses a transfer chain comprising a transposase recognition sequence, a label sequence, and a common sequence inserted at 5' ends of ds nucleic acid fragments [Claim 10], [column 74, lines 33-67])
(e) contacting the processed cells or cell nuclei of step (d) with the bead particles, wherein the nucleic acid fragments and the oligonucleotide molecules generate labeled nucleic acid molecules comprising the sequence of the nucleic acid fragments and the complementary sequence of the labeling sequence from the 5' end to the 3' end, or comprise the labeling sequence and the complementary sequence of the nucleic acid fragments. (e.g. Song discloses contacting nucleic acid templates with a bead substrate comprising an oligonucleotide probe having a hybridization domain that hybridizes to a complementary sequence on the template strand, follow by ligation of the template strand to the probeto generate label nucleic acid molecules. The resulting molecules comprise a nucleic acid fragment sequence and the complementary of the labeling probe. [column70, line 65-column 71, line 29][Figs. 7A- 7C])
However, Song’s tagmentation targets are dsDNA instead of RNA-cDNA hybrid resulting from reverse transcription of mRNA.
Chen discloses (b) providing multiple cells or cell nuclei comprising RNA; (c) performing processing on the RNA within the permeabilized cells or cell nuclei, wherein the processing comprises a reverse transcription step to form RNA-cDNA hybrid double-stranded nucleic acids; (d) incubating the RNA-cDNA hybrid double-stranded nucleic acids with a transposase complex comprising a transposase and a transposon sequence that the transposase is used to recognize and bind, and is capable of cleaving or breaking the RNA-cDNA hybrid double-stranded nucleic acids, (e.g. methods of labeling nuclear target-associated DNA using in-cell captured mRNA with intracellular tagmentation on DNA/RNA hybrid: permeabilizing a cell, transport the reverse transcriptase into the cells, then reverse transcribe mRNA to cDNA to form RNA-cDNA hybrid double-stranded nucleic acid. Add transpososomes into the cells and perform strand transfer reaction or tagmentation reaction on the RNA/cDNA hybrid inside the cells. [¶0007, 00018, 00049, and 00085].)
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Song’s in vivo tagmentation targets from dsDNA to RNA-cDNA hybrid resulting from reverse transcription of mRNA to enable capturing mRNAs inside of a cell is a more efficient way of capturing mRNA molecules and should allow for near complete mRNA capture. This will overcome the low mRNA capture efficiencies and high drop-out rate of conventional single cell capture method. This intracellular nucleic acid capture reaction dramatically simplifies the sample preparation workflow for single cell expression analysis, single cell genotyping and sequencing analysis, and will offer a cost-effective solution for single cell-based studies [Chen ¶00054].)
Regarding claims 25 and 26, Song discloses the sample comprise plurality of cells from animals, plants, or microorganisms, or any combination thereof. [column
25, lines 22-47]
Regarding claim 27, Chen discloses the RNA is reverse transcribed using a reverse transcriptase to form hybrid double-stranded nucleic acid comprising RNA and cDNA chains. [¶0007, 00018, 00049, and 00085]
Regarding claims 29 and 45, Chen discloses the nucleic acid fragment pool is used for constructing a transcriptome library or for transcriptome sequencing. (e.g. In-cell capture can also be used for whole transcriptome analysis [¶00099, 000106]
Regarding claim 30, Song discloses step (a), at least 2 of the processed cells or cell nuclei are provided, and/or, at least 2 beads are provided. [column 30, lines 31-33] [column 83, lines 19-20]
Regarding claim 31, Song discloses plurality of oligonucleotide can be immobilized on the synthetic particle (e.g. beads). One of ordinary skill would have understood that optimizing the number of oligonucleotide per bead (e.g. at least 10) is the matter of routine optimization base on desired capture efficiency, bead size, and reaction sensitivity. Accordingly, at least 10 oligonucleotide molecules on the bead is deemed prima facie obvious to a person of ordinary skill in the art.
Regarding claim 32, Song discloses the tag sequence comprises a second tag sequence and is selected from the following elements: a first amplification primer sequence, a second common sequence, a unique molecular tag sequence, a template switching sequence, or any combination thereof. (e.g. bead's second tag sequence [Fig 61, tagmented dsDNA's second tag sequence [column 90, lines 45-column 92, line 32])
Regarding claim 33, Song discloses contacting nucleic acid fragments to bead-immobilized oligonucleotides by (e1) lysing cells or cell nuclei [column 7, lines 33-34].
Regarding claim 34, Song and Chen discloses (i) generating multiple labeled nucleic acid molecules according to claim 24 and (ii) recovering and/or combining multiple labeled nucleic acid molecules to obtain a library of nucleic acid molecules. [Song column 42, lines 8-10][Chen ¶00014 and 00083]
Regarding claims 35 and 39, Song discloses enriching the labeled nucleic acid molecules.[column 25, line 31]
Regarding claim 36, Song discloses primer carrying a tag that can bind a partner molecule for enrichment [e.g. Biotin label in Fig 7B].
Regarding claim 37, Song discloses amplification of labeled nucleic acid molecules using adaptor-associated primer during library preparation [Fig. 13]. Song further teaches oligonucleotides comprising labeling molecules such as biotin that interact with binding partner such as streptavidin to enable capture and enrichment of nucleic acid products [Figs 7B and 7C]. Because affinity-labeled PCR primers routinely used to generate enrich amplification products via binding interactions, Song teaches or renders obvious performing amplification using first primer and/or the second labeled primers as recited in claim 37.
Regarding claim 38, Song discloses (iv) recovering and purifying the enriched products [column 51, lines 47-50].
Regarding claim 40, Chen discloses the target nucleic acid molecules comprise sequences encoding T-cell receptors (TCR) [00099].
Regarding claim 41, Song and Chen disclose a method for sequencing nucleic acids from cells or cell nuclei, comprising: constructing a nucleic acid library according to claim 34; and sequencing the nucleic acid library. [Song column 63, lines 17-28] [Chen 00013-00014]
Regarding claims 43 and 44, Song discloses a kit comprising: reverse transcriptase, transposase, at least 2 labeled transposome adaptors, where the transfer strand comprises common sequence, label sequence, and transposase recognition sequence barcoded beads[column 71, lines 30-54], where same barcode per bead and different barcode between beads (e.g. the bead is conjugated to a UMI [Fig 12] and "the cell label is identical for all barcodes attached to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads)." [column 31, lines 28-30]).
Song does not disclose transposase is capable of cleaving or breaking RNA-cDNA hybrid.
Chen discloses transposase is capable of cleaving or breaking RNA-cDNA hybrid [¶00067, 0007, 00018, 00049, and 00085]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Song’s kit to substitute dsDNA transposase with RNA-cDNA-hybrid transposase to enable tagmentation of RNA-cDNA hybrid. Chen explicitly discloses capturing mRNAs inside of a cell is a more efficient way of capturing mRNA molecules and should allow for near complete mRNA capture. This will overcome the low mRNA capture efficiencies and high drop-out rate of conventional single cell capture method. This intracellular nucleic acid capture reaction dramatically simplifies the sample preparation workflow for single cell expression analysis, single cell genotyping and sequencing analysis, and will offer a cost-effective solution for single cell-based studies [Chen ¶00054].
Song et al.
Claim(s) 42 is/are rejected under 35 U.S.C. 103 as being unpatentable over Song et al. (US11939622B2, EFD: 07/22/2019, of record).
Applicant’s assertion that Claim 42 is amended to depend on claim 24 is acknowledge. However, claim 42 directed to nucleic acid, thus the amended phrase “A nucleic acid library prepared according to the method of claim 24” constitutes a product-by-process limitation. "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) See MPEP § 2113.
Regarding claim 42, Song discloses a nucleic acid library comprising multiple nucleic acid molecules, wherein one strand of the nucleic acid molecules comprises a first common sequence, a first label sequence, a cDNA fragment sequence, a unique molecular tag sequence [Fig 12], a transposase recognition sequence (because Song uses adaptor-loaded Tn5 transposomes, the adaptor sequences necessarily include the claimed transposase recognition sequence), template switching sequence [column 14, line 47], a second label sequence, and a complementary sequence of a second common sequence [Fig 61, wherein the cDNA fragment comprises a sequence complementary to the 5' end sequence of RNA [column 43, lines 54-62]. The complementary sequences recite in claim 42 correspond to obvious complementary strands of adaptor elements disclosed by Song, and providing labels at both fragment ends or rearranging adaptor element order represent routine optimization design choice because such sequences are modular and interchangeable in sequencing library construct. Accordingly, the additional limitation in claim 42 is deemed prima facie obvious to a person of ordinary skill in the art. This reasoning is consistent with KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 - 97 (2007) (see MPEP § 2143,E)
Song et al., Chen et al., and Di et al.
Claim(s) 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Song et al. (US11939622B2, EFD: July 22nd 2019, of record) in view of Chen et al. (WO2020186094A1, EFD: March 12th 2020) and Di et al. (Proc Natl Acad Sci US A. 2020;117(6):2886-2893, of reccord).
Regarding claim 28, Chen discloses using Tn5 to tagment RNA-cDNA hybrids [¶00085, 000103 000106]. However, Chen does not disclose transposase complex is capable of randomly cleaving RNA-cDNA hybrids
Di discloses Tn5 transposase, which randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. prime the RNA/DNA heteroduplexes generated by reverse transcription [abstract].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to apply Di’s known Tn5 capable of randomly fragment RNA-DNA hybrid duplexes in the Song and Chen workflow because Di’s in-house pTXB1 Tn5 is shown to be 10-- 500-fold more effective than commercially available Tn5 transposomes (e.g. ATM and V50) [Discussion].
Song et al., Chen et al., and Zhu et al.
Claim(s) 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Song et al. (US11939622B2, EFD: July 22nd 2019, of record) in view of Chen et al. (WO2020186094A1, EFD: March 12th 2020) and Zhu et al. (Nat Struct Mo/ Biol 26, 1063-1070 (2019), of reccord)
Regarding claim 46, Song and Chen do not disclose dividing cells or nuclei into subsets and incubating each subset separately with transposase complexes having different first label sequences prior to combining subsets.
Zhu (Paired-Seq) discloses split-pool combinatory indexing in which cells or nuclei are partitioned into subsets, treated with indexed transposase adaptor complexes containing different barcode sequences, and subsequently pooled such that fragments from each subset contain distinct label sequences identifying their subset origin. [abstract]
As of the application' s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to combine these teachings to modify Song's method to incorporate Zhu's subset-specific indexed transposase labeling prior to pooling because spilt-pool indexing with different adaptor barcodes was a known strategy for increasing multiplex capacity and enabling identification of nucleic acid fragments from different cell populations in high-throughput single-cell sequencing workflow [Zhu's abstract]. Accordingly, the additional limitation in claim 46 is deemed prima facie obvious to a person of ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 - 97 (2007) (see MPEP § 2143, D).
Conclusion
No claims are 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 Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KHAI QUYNH TIEN PHAM/Examiner, Art Unit 1684
/JEREMY C FLINDERS/Primary Examiner, Art Unit 1684
