DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Status of the Application
Claims 46-63 are pending and under examination
The following Office Action is in response to Applicant's communication dated 05/06/2024.
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.
Claim(s) 55, 59, and 60 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 55, the phrase “the genomic DNA” lacks proper antecedent basis. However, claim 46, from which claim 55 depends, does not recite any genomic DNA. Hence, the phrase “the genomic DNA” lack antecedent basis.
Claim 59 recites “sample polynucleotides”, but the preceding claim recites polynucleotides from the cells are genomic DNA. It is unclear whether “sample polynucleotides” referring to either previously recited material or introduces distinct polynucleotides. For purposes of examination only, and to facilitate a complete analysis of the claim, the Examiner interprets “sample polynucleotides” as referring to previously recited material.
Claim(s) 60 depend from claim 59 and is/are therefore similarly rejected.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 61 rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Specifically, the claim is written in dependent from but fails to identify a previously set forth claim from which it depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
For purposes of examination only, and to facilitate a complete analysis of the claim, the Examiner interprets claim 61 to depends on claim 53 because claim 61 begins with “the genomic DNA” and claim 53 introduces genomic DNA. This interpretation is adopted solely for examination and does not resolve the lack of clarity in the claim language.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of pre-AIA 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a) the invention was known or used by others in this country, or patented or described in a printed publication in this or a foreign country, before the invention thereof by the applicant for a patent.
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of application for patent in the United States.
Claim(s) 46, 48, 49, 52, 53, 55, 61, and 63 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Linnarsson et al. (WO2010117620A2, EFD: March 23rd 2010).
Regarding claim 46, 52, 55 Linnarsson discloses a method of analyzing cells, the method comprising, separating individual cells into unique partitions comprising uniquely-barcoded adaptor oligonucleotides; generate barcoded polynucleotides; attaching the uniquely-barcodes adaptor oligonucleotides to polynucleotides from the cells to form barcoded polynucleotides; (e.g. single cells are placed in separate wells of a 96-well plate and lysed; tagged reverse transcription is performed on each sample to produce cDNA [page 4, lines 10-13]. Tags act as a "'barcode" to identify which single cell from each cDNA sample originated. Thus, each cDNA sample can be linked to a single cell, even after the tagged cDNA samples have been pooled in the method. [page 19, lines 4-11])
pooling the barcoded polynucleotides from partitions; (e.g. cDNA samples are pooled and amplified[page 4, lines 10-13])
sequencing the barcoded polynucleotides. (e.g. sequencing is performed to obtain 100 million reads; and (F) identification of expressed genes and identification of cells from which they originated. [page 4, lines 10-13])
Regarding claim 48, Linnarsson discloses the polynucleotides from the cells are reverse transcribed mRNA (cDNA). (e.g. single cells are placed in separate wells of a 96-well plate and lysed; tagged reverse transcription is performed on each sample to produce cDNA [page 4, lines 10-13].)
Regarding claim 49, Linnarsson discloses reverse transcribing mRNA from the cells in the partitions with partition-specific barcoded primers to form barcoded cDNAs. (e.g. Figure 4 shows an example of a cDNA synthesis primer (CDS) comprising a 5' amplification primer sequence (APS), a cell tag and a 3' RNA complementary sequence (RCS).)
Regarding claim 53, Linnarsson discloses the polynucleotides from the cells are genomic DNA. (e.g. If the polynucleotide is derived from genomic DNA, expression can include splicing of the m&~A in an eukaryotic cell. [page 22, lines 3-4].)
Regarding claim 61, Linnarsson discloses the genomic DNA is treated with bisulfite, converting unmethylated cytosines to uracil. [column 56, line 35]
Regarding claim 63, Linnarsson discloses the attaching comprises amplifying the polynucleotides from the cells with the uniquely-barcodes adaptor oligonucleotides. (e.g. tagging the individual cDNA, pooling the tagged cDNA samples and amplifying the pooled cDNA samples to generate a cDNA library. [abstract]).
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 pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
Linnarsson et al.and Drmanac et al.
Claims 57, 59, 60, and 62 is/are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Linnarsson et al. (WO2010117620A2, EFD: March 23rd 2010) in view of Drmanac et al. (US8592150B2, Filed June 15th 2010)
Regarding claim 57, Linnarsson does not disclose the partitions comprise methyl-sensitive endonucleases.
Drmanac discloses methods for preparing sequencing library by placing tags/adaptors in each emulsion droplets. And the methods and compositions of invention can be used for genomic methylation analysis. The most economically accessible method involves bisulfate treatment of genomic DNA and sequencing of repetitive elements or a fraction of the genome obtained by methylation specific restriction enzyme fragmenting [column 55, lines 60-67].
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 include the methylation specific restriction enzyme within the genomic DNA containing partitions to preserve association between resulting fragments and partition specific sample/barcode, enable direct detection of specific epigenetic states. Additionally, Drmanac teaches that this is a economically accessible method for processing genomic DNA. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Regarding claim 59, Linnarsson and Drmanac discloses merging partitions comprising uniquely barcoded adaptors with partitions comprising sample polynucleotides. (e.g. Linnarsson discloses cDNA samples are pooled and amplified [page 4, lines 10-13]. Drmanac discloses droplets containing nucleic acid fragments are combined with emulsion droplets containing tags. The combined droplets provide an enclosed space for ligation of tags to fragments, such that different combined droplets contain fragments tagged with different tags. [column 92, lines 18-35])
Regarding claim 60, Drmanac discloses barcode ligation and methyl-sensitive endonucleases digestion. However, Drmanac does not explicitly disclose the order of the steps. 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 ligate the barcode either before or after digestion because selecting the order of adaptor ligation and enzymatic digestion would have been the matter of routine optimization to achieve predictable result of generating barcoded DNA fragments suitable for specific chosen downstream sequencing and methylation analysis. See MPEP § 2144.05 (II) "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 62, Linnarsson does not disclose the attaching comprises ligating the uniquely-barcodes adaptor oligonucleotides to the polynucleotides from the cells.
Drmanac discloses methods for preparing sequencing library by placing tags/adaptors in each emulsion droplets. Then, droplets containing nucleic acid fragments are combined with emulsion droplets containing tags. The combined droplets provide an enclosed space for ligation of tags to fragments, such that different combined droplets contain fragments tagged with different tags. [column 92, lines 18-35]. Drmanac further discloses tags may contain “barcode” sequence [column 48, line 41] and target nucleic acids maybe cDNA [column 80, line 52].
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 incorporate Drmanac’s unique adaptor barcode to Linnarsson’s cDNA post reverses transcription because adaptor ligation is a well-known and conventional method to prepare cDNA libraries for next-gen sequencing. Both techniques uniquely label cDNA for pooled multiplex sequencing. Substituting one known barcoding approach for another would have yielded the predictable result of identifiable cDNA molecules and enable a POSITA to trace the analyte to cell of origin. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Linnarsson et al. and Brouzes et al.
Claim 47 is/are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Linnarsson et al. (WO2010117620A2, EFD: March 23rd 2010) in view of Brouzes et al. (Proc. Natl. Acad. Sci. U.S.A. (2009) 106 (34) 14195-14200)
Regarding claim 47, Linnarsson does not discloses the single cell partitions are droplets. Drmanac discloses aliquots emulsion droplets of the nucleic acids, not cells.
Brouzes discloses droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells, which allows for the encapsulation of single cells and reagents in independent aqueous microdroplets. Brouzes describes droplets as discrete reactors and teaches the platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses [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 perform Linnarsson’s single cell barcoding reactions in Brouzes’ droplet partitions because droplet-based technique enable physical and chemical isolation of droplets eliminating the risk of cross-contamination, the fast and efficient mixing of the reagents that occurs inside droplets, the ability to digitally manipulate droplets at a very high-throughput [page 14195]. The substitution of droplet over Linnarsson’s 96 well plates would have predictably provided physical isolation of individual cells and their respective barcoding reactions while reducing reaction volume and allow parallel processing. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Linnarsson et al. and Xie et al.
Claim 54 is/are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Linnarsson et al. (WO2010117620A2, EFD: March 23rd 2010) in view of Xie et al. (BMC Bioinformatics 10, 80 (2009).)
Regarding claims 54, Linnarsson does not discloses determining copy number variation (CNV) from the sequencing.
Xie discloses a method to detect copy number variation using shotgun sequencing, CNV-seq. The method is based on a robust statistical model that allows confidence assessment of observed copy number ratios with high specificity (91.7 – 99.9%) and sensitivity (72.2 – 96.5%). Xie further discloses that the number of reads, not the length of the reads is the key factor determining the resolution of detection, meaning the method works well with next-generation sequencing data output, which produces large number of short reads [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 analyze the sequencing result generated by Linnarsson and Drmanac using Xie’s CNV-seq because by utilizing a robust statistical model and emphasizing read count volume over length, the data analysis approach successfully maximizes sensitivity and specificity, even at low sequencing coverages, making it compatible with both microarray methods and NGS data output [Conclusion]. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Linnarsson et al., Brouzes et al., and Eshoo et al.
Claim 56 is/are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Linnarsson et al. (WO2010117620A2, EFD: March 23rd 2010) in view of Brouzes et al. (Proc. Natl. Acad. Sci. U.S.A. 106 (34) 14195-14200) and Eshoo et al. (US9890408B2, EFD: Oct 15th 2010)
Regarding claim 56, Linnarsson and Brouzes do not discloses multiple-displacement amplification (MDA) is performed within a droplet on a cell's genome, prior to fragmentation and adaptor ligation.
Eshoo teaches whole genome amplification by multiple displacement amplification using random oligonucleotide primers and a stand-displacing polymerase, such as Phi29 [column 7, lines 38-49]. Eshoo further teaches method for amplifying a nucleic acid is carried out by contacting said nucleic acid with the reaction mixture in the form of an emulsion which may be prepared using a detergent and a hydrophobic polymer. Upon completion of the amplification reaction, the emulsion was broken and DNA is recovered for further analysis, including sequencing using next generation sequencing methods [columns 3-5]. Eshoo explains the DNA of the sample may include an entire genome [column 4, lines 16-17] and MDA is useful for amplifying small quantities of DNA [column 10, lines 19-20].
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 perform Eshoo’s emulsion MDA reaction within individual cell droplets because emulsion MDA reportedly able to amplifying an entire genome from small quantities of sample DNA for downstream sequencing analysis [column 9 line 64-column 10 line 13]. Additionally, it would have been obvious to amplify small amount of whole genome DNA before fragmentation and adapter ligation to ensure appropriate amount is available for subsequence sequencing library preparation. A skilled artisan would have had a reasonable expectation of success because Eshoo expressly teaches recovering emulsion MDA products for downstream next generation sequencing, and Brouzes teaches cellular materials and reagents can be maintained and react within the droplets. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Linnarsson et al. Drmanac et al., and Zheng et al.
Claim 58 is/are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Linnarsson et al. (WO2010117620A2, EFD: March 23rd 2010) in view of Drmanac et al. (US8592150B2, Filed June 15th 2010), and Zheng et al. (Nucleic Acids Res. 38.16 (2010): 5527-5534.).
Regarding claim 58, Drmanac does not explicitly explains the disclosed methylation specific restriction enzyme fragmenting digest methylated sites. However, as of the application’ s effective filing date, it is known in the art that DNA methylation-dependent restriction endonuclease, such as MspJI, recognizes methylated cytosines and digests at the location of the modification on the genomic DNA [Zheng et al., Abstract]. Accordingly, it would have been prima facie obvious to a person of ordinary skill in the art to use methyl-sensitive endonucleases such as MspJI to digest genomic DNA at methylated sites. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Allowable Subject Matter
Claims 50 and 51 contain subject matter free of prior arts, but objected to as being dependent upon a rejected base claim. The following references were considered as the closest prior arts:
Linnarsson and Drmanac do not disclose or suggest multicompartment droplet design (inner and outer droplet, wherein heat activated bursting of inner droplet enable transcriptase to contact the mRNA) recited in claim 50 and 51.
Sun et al. (ACS Appl. Mater. Interfaces 2010, 2, 12, 3411–3416) was considered for claims 50 and 51 because the reference teaches a microfluidic melt emulsification method for encapsulation and release of actives is presented. Actives encapsulated inside the solid shell can be controllably and rapidly released by applying a temperature trigger to melt the shell. The reference further teaches encapsulate multiple actives in multicompartment capsules, which can be use as multifunctional microreactors [Abstract]. However, Sun does not teach nor suggest that such design is biocompatible and can be used in cell encapsulation, reverse transcription reactions, or sequencing library preparation. Incorporating Sun’s controlled release microcapsule system for Linnarsson and Drmanac’s gene analysis reactions while preserving optimal reaction environment present significant technical difficulty and would not have been predictable or routine modification.
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.
US 9,347,059 B2
Claim(s) 46, 59, and 62 rejected on the ground of nonstatutory double patenting as being unpatentable over claim(s) 1, 2, 11, 13, and 20 of U.S. Patent No. 9347059B2 (the '059 patent). Although the claims at issue are not identical, they are not patentably distinct from each other because the rejected claims of the present invention would be anticipated and/or rendered obvious by the subject matter in the claims of the reference patent.
Regarding present claim(s) 46, the claim of the '059 patent discloses method of analyzing cells, the method comprising, separating individual cells into unique partitions comprising uniquely-barcoded adaptor oligonucleotides; attaching the uniquely-barcodes adaptor oligonucleotides to polynucleotides from the cells to form barcoded polynucleotides; pooling the barcoded polynucleotides from partitions; and sequencing the barcoded polynucleotides. (e.g. as per claim(s) 1, 11, and 13 of the ‘059 patent)
Regarding present claim(s) 59, the claim of the '059 patent discloses merging partitions comprising uniquely barcoded adaptors with partitions comprising sample polynucleotides. (e.g. as per claim(s) 1 and 2 of the ‘059 patent)
Regarding present claim(s) 62, the claim of the '059 patent discloses the attaching comprises ligating the uniquely-barcodes adaptor oligonucleotides to the polynucleotides from the cells. (e.g. as per claim(s) 20 of the ‘059 patent)
Conclusion
No claims are allowed
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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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
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