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 Claims
Claim(s) 1-8, 13, 21, 24-28, 31, 34-35, 38, and 41 are currently pending and examined here.
The following Office Action is in response to Applicant's communication dated 06/20/2024.
Claim Rejections - 35 USC § 112(b)
Claim(s) 21 and 38 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 21 recites “The method of claim 4, wherein the lysing step (e)”, but claim 4 identifies lysis step as step (f). Step (e) recites “optionally amplifying one or more biomolecules under conditions to increase copy number of said one or more biomolecules”.
Claim 38 recites “The method of claim 25, wherein the lysing step (e)”, but claim 25 identifies lysis step as step (d). There is no step (e) in claim 25.
Claim Objections
Claim 31 recites “under conditions that formation of permeable” is grammatically incomplete. The claim appears to intend “under conditions that allow/permit formation of permeable”.
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 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)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-3, 24-28, 31, 34-35, 38, and 41 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Lan et al. (WO2018119301A1, disclosed in IDS).
Regarding claim 1-3, Lan discloses a method of sequencing one or more nucleic acids from a single cell comprising the steps of: (a) preparing single cells for coding comprising compartmentalizing a population of cells in permeable compartments comprising single cells; (e.g. a population of single cells may be flowed through a device, under conditions sufficient to effect inertial ordering of the cells in the channel, thereby providing periodic injection of the cells into the droplet generator to encapsulate single cells in individual droplets. [¶0053 and Fig. 2]. Droplets are hydrogel microspheres (microgels), which are permeable to molecules with hydraulic diameters smaller than the pore size, including enzymes, detergents, and small molecules, but sterically trap large molecules such as genomes. [¶0054-0056 and ¶00156])
(b) barcoding nucleic acid molecules associated with the single cells, wherein a unique barcode is used for each single cell; (e.g. the cells are lysed, genomes are fragmented, and unique barcodes are attached to all fragments [¶0062 and Fig. 2])
(c) sequencing one or more nucleic acids from a single cell.[¶0060-0061]
Regarding claims 24-28, 31, 34-35, and 38 are rejected for the same reason as claim 2 because claim 2 recites alternative compartmentalizing methods, and claims 24-28, 31, 34-35, and 38 further limit certain alternatives without excluding the hydrogel particles option. Thus, Lan disclosure of hydrogel compartmentalizing method satisfies the method encompasses by claim 2 and applies to claims 24-28, 31, 34-35, and 38.
Regarding claim 41, Lan further discloses cells are selected from mammalian cells, bacterial cells, fungal cells, yeast cells, and plant cells. [¶0061-0062].
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.
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.
Lan et al and Li et al.
Claim(s) 4, 7-8, and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lan et al. (WO2018119301A1, disclosed in IDS) in view of Li et al. (LOC 18.17 (2018): 2575-2582).
Regarding claim 4, Lan further discloses the preparing single cells for barcoding comprises: (a) preparing a suspension comprising a population of single cells; (b) encapsulating the population of single cells in gel droplets to provide a population of gel droplets, wherein each gel droplet contains a single cell; (e.g. a population of single cells may be flowed through a device, under conditions sufficient to effect inertial ordering of the cells in the channel, thereby providing periodic injection of the cells into the droplet generator to encapsulate single cells in individual droplets. [¶0053 and Fig. 2]. In some embodiments, a droplet in which a single cell is encapsulated comprises a polymeric material. For example, suitable polymeric materials may include interpenetrating polymer networks (TPNs); a synthetic hydrogel [¶0054])
(c) polymerizing the population of gel droplets to provide a population of polymerized gel droplets; (e.g. reference discloses single cells can be encapsulated in molten gel droplets can be solidified into solidified microgel droplets by cooling [¶0058], chemically crosslinking, photo-crosslinking [¶00137]. This disclosure encompasses “polymerizing” limitation since Applicant’s specification define “polymerizing of step (c) comprises one or more of cooling, chemical crosslinking, photo-crosslinking, and ionic interaction crosslinking.”[applicant’s specification ¶0012])
(e) optionally amplifying one or more biomolecules under conditions to increase copy number of said one or more biomolecules; and (e.g. target gene fragments are amplified [¶0088-00108])
(f) lysing the single cells within the population of gel droplets under conditions that allow cell lysis. (e.g. cells are lysed in bulk, while maintaining isolation of genomic DNA from different cells in different solidified microgels [¶0062-0070])
Lan does disclose the operating devices and systems may include sorter for sorting droplets, into one or more flow channels. The droplets are sorted base one or more characteristics of the droplets including composition, size, shape, buoyancy, or other characteristics.[¶00123]. However, Lan does not disclose b) encapsulating the population in a polydispersed emulsion and c) separating the polymerized gel droplets by size under conditions that allow selection of a population of gel droplets that each comprise a single cell” as an explicit step in the method.
Li discloses monodisperse emulsions generated by microfluidics can be limited, , especially for very viscous hydrogel solutions and following reaction or culture droplet size can fluctuate in a reaction-dependent fashion. To solve this limitation, polydisperse manufacture of hydrogel droplets can be generated more rapidly using both continuous microfluidic and traditional batch emulsion generation approaches. Li further discloses high-throughput sorting of hydrogel droplets by size, enable the selection of droplets having a specific size from complex mixtures following reaction or culture [Introduction]. Li also mentioned the disclosed size sorting method follows Poisson statistic and able to separate droplets containing single cells from different subpopulations [pages 2579-2580].
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 Lan’s hydrogel microgel single cell sequencing workflow to include Li’s polydisperse hydrogel droplets and an extra step of size sorting because Li identifies a practical limitation in monodisperse hydrogel droplet workflow: droplet generation rate is limited, especially for viscous hydrogel solutions, and droplet size can fluctuate after reaction or culture, while polydisperse droplet generation, including traditional batch emulsion approaches, is faster. Li further teaches that size sorting of polydisperse hydrogel emulsions can purify the droplets toward monodispersity, select droplets of defined size from complex mixtures, while maintaining cell viability [abstract and introduction]. A skilled artisan therefore would have been motivated to apply Li’s high-throughput size sorting method to Lan’s hydrogel sing-cell sequencing workflow to enrich/select a desired population of polymerized single cell gel droplets before downstream cell lysis, barcoding, and sequencing, with reasonable expectation of success because both references concern hydrogel droplets for cell encapsulation. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143)
Regarding claim 7, Lan further discloses the preparing comprises the amplifying of step (e), and wherein said amplifying comprises an amplification technique multiple displacement amplification (MDA) [¶0083].
Regarding claim 8, claim 4 recites step (e) as optional amplification, and claim 8 specify that when performed, the optimally amplification occurs before size separation step (d). Lan discloses amplification of target gene fragments in hydrogel single cell workflow. Li teaches that hydrogel droplet size can fluctuate after reaction or culture and size sorting is useful for selecting droplets of specific size from complex mixtures following reaction or culture [introduction]. Thus, it would be obvious to perform Lan’s step (e) before size sorting step (d), so that the target post amplification droplet population could be accurately enriched before further downstream processing.
Regarding claim 21, Lan further discloses the lysing step (f) further comprises processing one or more biomolecules from the lysed cells, wherein said biomolecules comprise nucleic acids and/or proteins, and wherein said processing comprises digesting [¶0066], labelling [¶0080-00108], capturing and conjugating said biomolecules (e.g. conjugated with biotin (or other biomolecule) to facilitate downstream capture of barcoded DNA fragments using streptavidin-coated beads[¶0097]).
Lan et al, Li et al. and Hatori et al.
Claim(s) 5-6 and 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lan et al. (WO2018119301A1, disclosed in IDS) in view of Li et al. (LOC 18.17 (2018): 2575-2582) and Hatori et al. (Anal. Chem. 2018, 90, 16, 9813–9820, disclosed in IDS).
Regarding claim 5, Li recognizes that hydrogel droplet can also be manufactured more rapidly using traditional batch emulsification. A person of ordinary skill in the art would have known vortex emulsification to be one of the said traditional batch emulsification approaches.
Hatori further confirms this by teaching particle-templated emulsification (PTE), an approach for generating compartmentalized reactions in droplets by mixing hydrogel particles, sample solution, and oil, then emulsifying the mixture by vortexing [abstract and introduction]. Hatori explains that the complex microfluidic devices require specialized microfluidic hardware and skill, making the technology difficult to translate to some biology labs, hence creating major inefficiency in microfluidics research. PTE provides a simpler and more affordable way to generate droplet compartments without microfluidic systems. Hatori also demonstrates under controlled concentration, PTE is capable of encapsulate single yeast cell per droplet [“PTE for Single Cell Biology” section].
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 Lan and Li’s hydrogel single cell sequencing workflow to prepare the cell suspension and encapsulate the cells using Hatori’s microfluidics-free vortex emulsification approach because Li expressly identifies traditional batch emulsion generation as one of faster alternative for producing hydrogel droplets and Hatori provides a predictable vortex-based batch emulsification method that avoids costly specialized microfluidic equipment while remaining compatible with biological reactions and single-cell encapsulation. The modification would allow implementation, reduce reliance on specialized equipment, and facilitate droplet compartmentalization in ordinary biological laboratory while still producing cell containing hydrogel droplets for downstream processing, size sorting, barcoding, and sequencing. A skilled artisan would have reasonable expectation of success because Lan, Li, and Hatori each concern hydrogel droplets used to compartmentalize cells. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Regarding claim 6, Hatori further discloses one or more of the steps of polymerizing in step (c), separating in step (d), optionally the amplifying in step (e), lysing in step (f), and barcoding are optionally not performed using a microfluidic device. (e.g. Fig. 1f shows target sequences are amplified in the droplets not performed using a microfluidic device. Hatori further discloses components such as barcoded oligonucleotides required for single cell transcriptome sequencing, or reagents necessary for cell lysis or reporter assays can also be introduce to the droplets using disclosed PTE method [introduction]).
Regarding claim 13, Li discloses generating encapsulating cells comprises adding an immiscible carrier and allow formation of a polydispersed emulsion. (e.g. hydrogel droplets of different sizes are generated using Novec™ 7500 fluid as continuous phase, gelatin solution was used as dispersed phase (support oil/aqueous emulsion system) [experimental section]). However, Li’s droplets are made using a 3D-printed droplet generator, not by “agitating”.
As mentioned above, Hatori discloses PTE, an approach for generating compartmentalized reactions in droplets by mixing hydrogel particles, sample solution, and oil, then emulsifying the mixture by vortexing [abstract and introduction]. Vortex is one of the agitating methods listed in Applicant’s specification ¶0010. Hence, Hatori discloses this limitation.
The rationale for combining the Lan, Li, and Hatori references with respect to claim 13 is the same as set forth above for claim 5 and is incorporated herein by reference, as claim 13 does not introduce a limitation that would alter the motivation to combine or the predictable resulted achieved by the combination.
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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