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 .
Priority
This application is a 371 of PCT/EP2022/084074 filed 12/01/2022, which claims benefit of U.S. Provisional Application No. 63/284,669, filed 12/01/2021.
Status of the Claims
Claims 51-76 are currently pending and under examination. Claims 1-50 are canceled.
Claim Objections
Claim 64 is objected to because of the following informalities: Claim 64 recites two commas within a limitation “The method of claim 51, further comprising, , generating”. Appropriate correction of this grammatical or spelling error is required.
Claim 76 is objected to for minor informalities: The preamble of claim 76 recites the limitation of “nucleic acid within in a droplet” which contains a spelling/grammar error.
Claim 76 also recites the limitation “(3) comprising a carrier oil”, which contains a redundant word “comprising”. Amendment of claim 76 to replace the noted limitation above with “3)
Specification
The disclosure is objected to because of the following informalities: the specification is objected to for disclosing sequences with improper sequence nomenclature on pages 31-32, 36-38, as it relates to the sequence identifiers assigned to them in the sequence listing as required by 37 CFR 1.821 (d).
Please use the underlined nomenclature “SEQ ID NO: X” or “SEQ ID NO. X” to properly disclose P5 primer consisting SEQ ID NO: 1 on page 31 and TSO primer consisting SEQ ID NO: 2 on page 31 and on page 37. A request for proper disclosure of SEQ ID NO: 5 within table 1 on page 36 and within Table 5 on pg 41 is requested as well as SEQ ID NOS: 3-9 on page 38 and SEQ ID NOS: 10-11 on page 42. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 52, 53 and 69-76 are 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.
Regarding claim 52, the phrase "preferably" followed by the limitation of “20 to 150 nucleotides in length” as well as the term “more preferably” followed by the limitation “30 to 120 nucleotides in length”, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Specifically, it is not known if the embodiments that follow the words “preferably” or “more preferably” are required or suggested/optional limitations in the claim.
Regarding claim 53(i), the phrase "preferably" followed by the limitation of, “wherein the UMI is a random nucleotide sequence 4 to 50 nucleotides in length” as well as the term “more preferably” followed by the limitation “in the range of 4-16 nucleotides in length”, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Specifically, it is not known if the embodiments that follow the words “preferably” or “more preferably” are required or suggested/optional limitations in the claim.
Regarding claim 53(ii), the phrase "preferably" followed by the limitation of, “longer than 4 but shorter than 100 nucleotides” and phrase "preferably" followed by the limitation “6 to 70 nucleotides in length”, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Specifically, it is not known if the embodiments that follow the words “preferably” or “more preferably” are required or suggested/optional limitations in the claim.
Regarding claim 69, the phrase "preferably" followed by the limitation of, “between 10-500 µm” and phrase "more preferably" followed by the limitation “between 20-100 µm”, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). It is not known if the embodiments that follow the words “preferably” or “more preferably” are required or suggested/optional limitations in the claim.
Regarding claim 70, the phrase "preferably" followed by the limitation of, “a volume of about 0.1 - 10 nanoliters” and phrase "more preferably" followed by the limitation “a volume of about 0.1 to 3 nanoliters”, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). It is not known if the embodiments that follow the words “preferably” or “more preferably” are required or suggested/optional limitations in the claim.
Regarding claim 71, the phrase "preferably" followed by the limitation of, “wherein the hydrogel beads have a size in the range of 1-200 µm” and phrase "more preferably" followed by the limitation “in the range of 10-80 µm”, and the limitation “in the range of 20-70 µm” renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Specifically, it is not known if the embodiments that follow the words “preferably” or “more preferably” are required or suggested/optional limitations in the claim.
Regarding claim 72(d), the phrase "preferably" followed by the limitation of, “at least 10%” and phrase "more preferably" followed by the limitation “at least 50%”, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Specifically, it is not known if the embodiments that follow the words “preferably” or “more preferably” are required or suggested/optional limitations in the claim.
Regarding claim 76(1), the phrase "preferably" followed by the limitation of, “the molecular tag of one particle is different from the molecular tags of other particles”, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claim 76, the limitation of step (5) i.e. “a second microfluidic chip configured to produce the further water-in-oil droplet from which the microcapsule is produced”. This limitation lacks clarity and thus, is indefinite. It is unclear whether step (5) is drawn to an optional step of providing a second microfluidic chip within a first microchip for generating water-in-oil droplet, wherein the second microfluidic chip produces a semi-permeable microcapsule by an aqueous phase separation to generate an additional water-in-oil droplet.
Claims 73-75 depend from claim 70 and are indefinite because these claims recites limitations that directed to step(s)(c) and (d). It is noted here that step (c) or (d) are not found in/recited by claim 70, but rather claim 72.
Claim Rejections - 35 USC § 112
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 56 is 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. Claim 56 depends from claim 51 but recites the limitation, "may comprise".
This limitation encompass an absence of the elements after the word “comprise”, rendering claim 56 s a claim that is not distinct from claim 51. An amendment of “may comprise” to “comprising” is suggested.
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.
Claims 51-76 are rejected under 35 U.S.C. 103 as being unpatentable over Pellegrino et al. (2018, Genome research, 28(9), pp.1345-1352) as evidenced by Zilionis et al., (2017, Nature protocols, 12(1), pp.44-73), and further in view of Weitz et al. (pub. October 29, 2015, WO2015/164212A1) and Polansky (US2004/0023207).
Regarding claims 51-61 and 63-64 and 66-67 and 72, Pellegrino et al. teach a novel two-step microfluidic droplet workflow that enables efficient and massively parallel single-cell PCR-based barcoding. The microfluidic workflow first encapsulates individual cells in droplets, lyses the cells, and prepares the lysate for genomic DNA amplification using proteases. Following this lysate preparation step, the proteases are inactivated via heat denaturation, and droplets containing the genomes of individual cells are paired with cell-identifying barcodes and PCR amplification reagents (pg 1345, right col., section entitled Results).
Pellegrino et al. teach droplets containing cell lysate (i.e. the instant microcapsule comprising a core, a semi-permeable shell, and nucleic acid(s) obtained from a biological species, wherein the nucleic acid is comprised in the core).
Specifically, Pellegrino et al. teach that cells are first encapsulated with lysis buffer containing protease and incubated to promote proteolysis. Protease activity is then thermally inactivated (see pg 1346, the legend of Fig. 1B and see also pg 1350., right col., section entitled “Cell encapsulation and droplet PCR”). Accordingly, Pellegrino et al. teach prior to co-encapsulation, the nucleic acid(s) are generated inside the plurality of microcapsules.
Pellegrino et al. also teach generation of barcode bead droplets (i.e. hydrogel bead particle, or the instant particle comprising a molecular tag).
Specifically, Pellegrino et al. synthesized hydrogel beads with oligonucleotides containing both cell-identifying barcodes and different gene specific primer sequences (pg 1346, left col., 2nd para and pg 1350, left col., section entitled Generation of barcode containing beads and pg 1350, right col., 1st para).
Pellegrino et al. teach co-encapsulating in a plurality of droplets, (i) a plurality of microcapsules (containing cell lysate comprising nucleic acids) and (ii) a plurality of particles comprising a molecular tag.
Pellegrino et al. teach that the barcoded (hydrogel) beads were microfluidically combined with droplets containing cell lysate generated with or without the protease reagent (pg 1346, left col., 2nd para). Pellegrino et al. teach for the plurality of droplets generated for co-encapsulation, on average, a droplet comprise one semi-permeable microcapsule that contains nucleic acid in its core and one barcoded (hydrogel) bead particle.
Pellegrino et al. teach the droplets containing the cell lysate are paired and merged with droplets containing PCR reagents and molecular barcode-carrying hydrogel beads (see pg 1346, the legend of Fig. 1B and see also pg 1350., right col., section entitled “Cell encapsulation and droplet PCR”). Pellegrino et al. teach the merge of droplets containing the cell lysate with the PCR reagent/barcode bead droplets was performed using a microfluidic electrode (pg 1350, left col., section entitled Fabrication and operation of microfluidic devices).
Pellegrino et al. teach a method that comprises release of the molecular tag from the particle and/or releasing the nucleic acid from the microcapsule (see pg 1346, left col., 2nd para)
Pellegrino et al. particularly teach that prior to PCR amplification, the oligonucleotides were photo-released from the hydrogel supports with UV exposure (pg 1346, left col., 2nd para).
Pellegrino et al. teach attaching of molecular tag released from hydrogel bead particle to the nucleic acid in the droplet via droplet PCR (pg 1350, right col., section entitled Cell encapsulation and droplet PCR).
Regarding claim 63, Pellegrino et al. teach the molecular tag is covalently attached to the particle and at least one of the molecular tags comprises a cleavable linker selected from one of photocleavable linker, chemically cleavable linker, or enzymatically cleavable linker (pg 1350, all text of section entitled Generation of barcode containing beads: wherein Pellegrino et al. teach acrydite modified barcode oligonucleotide attached to hydrogel and a photocleavable linker).
Regarding claim 65, Pellegrino et al. teach treating the semi-permeable microcapsules comprising the lysates in order to deplete one or more enzymatic inhibitors present in the lysate (see pg 1350, right col., section entitled “Cell encapsulation and droplet PCR”: see use of RNAse A).
Regarding claims 65 and 73, Pellegrino et al. also teach (ii) amplifying the nucleic acids in the microcapsules using a DNA polymerase driven reaction; and/or (iv) attaching the oligonucleotides to the fragmented nucleic acids (pg 1350, right col., section entitled “Cell encapsulation and droplet PCR”).
Pellegrino et al. teach amplifying the nucleic acids using a DNA polymerase driven reaction following step c) which relates to treating lysate containing microcapsules to purify the released nucleic acids and/or deplete one or more enzymatic inhibitors present in the lysates and before step (d) which relates to producing a plurality of microfluidic droplets using a microfluidic device.
Pellegrino et al. teach amplifying the barcoded nucleic acids using DNA polymerase, after producing barcoded nucleic acids, where the nucleic acids from each cell comprise the same cell barcode which is different from the cell barcode of nucleic acids from other cells in other droplets; and prior to releasing the barcoded nucleic acids by breaking the plurality of droplets (pg 1350, right col., section entitled “Cell encapsulation and droplet PCR”).
Regarding claim 68, Pellegrino et al. teach each particle of the plurality of particles comprises a molecular tag with a unique cell barcode (via citing Zilionis et al., (2017, Nature protocols, 12(1), pp.44-
Regarding claim 76, Pellegrino et al. teach the following claimed components:
(1) one or more particles comprising a molecular tag, wherein the molecular tag is a barcoding DNA oligonucleotide, and the molecular tag of one particle is different from the molecular tags of other particles;
(2) one or more reagents in aqueous solution suitable for creating the water-in-oil droplet;
(3) a carrier oil supplemented with a surfactant, wherein the surfactant is suitable to stabilize the water-in-oil droplets and prevent against coalescence, and
(4) either:
(i) one or more microcapsules, each microcapsule having a core, a semi- permeable shell and a biological sample comprising a nucleic acid comprised in the core; or
(ii) a shell precursor and a core precursor, suitable for making a semi-permeable microcapsule by aqueous phase separation in a further water-in-oil droplet.
(5) a microfluidic chip configured to produce the water-in-oil droplet.
Omitted from Pellegrino et al. (claims 69-71, 74-76)
Regarding claim 69, Pellegrino et al. do not teach microcapsules with average diameter of at least about 1 µm, or between 10-500 µm, or between 20-100 µm.
Regarding claim 70, Pellegrino et al. do not teach droplets with a volume of less than about 100 nanoliters, about 0.1 - 10 nanoliters, or a volume of about 0.1 to 3 nanoliters.
Regarding claim 71, Pellegrino et al. do not teach hydrogel beads having a size in the range of 1-200 µm or in the range of 10-80 µm, or in the range of 20-70 µm.
Regarding claim 74, Pellegrino et al. do not teach converting released RNA to cDNA in the semi-permeable microcapsule using a reverse transcription reaction following step c) and before step d).
Regarding claim 75, Pellegrino et al. do not teach fragmenting the nucleic acids in the microcapsules using chemical, enzymatic or physical means following step c) and before step d).
Regarding claim 76, Pellegrino et al. do not teach a kit.
Zilionis et al. (claims 69, 71 and 74-75)
Regarding claim 69, Zilionis et al. teach microcapsules with average diameter of at least about 1 µm, or between 10-500 µm, or between 20-100 µm (see pg 53, para 12: wherein disclosed are Droplet diameter in the 58- to 63-µm range, ideally 60 µm).
Regarding claim 71, Zilionis et al. teach hydrogel beads having a size in the range of 1-200 µm, or in the range of 10-80 µm, or in the range of 20-70 µm (see pg 53, para 11: wherein disclosed are hydrogel droplets of 125 pl (62 µm diameter)).
Regarding claim 74, Zilionis et al. teach converting the released RNA to cDNA in the semi-permeable microcapsule using a reverse transcription reaction following step c) and before step d)(see abstract).
Regarding claim 75, Zilionis et al. teach fragmenting the nucleic acids in the microcapsules using chemical, enzymatic or physical means following step c) and before step d).
Weitz et al. (claims 69-71)
Regarding claim 69, Weitz et al. teach microcapsules with average diameter of at least about 1 µm, or between 10-500 µm, or between 20-100 µm (see pg 23, lines 5-13 and pg 92, claim 5, wherein the plurality of microfluidic droplets have an average diameter of less than about 1 mm).
Regarding claim 70, Weitz et al. teach droplets with a volume of less than about 100 nanoliters, about 0.1 - 10 nanoliters, or a volume of about 0.1 to 3 nanoliters (pg 45, lines 31-33 and pg 92, claim 57: microfluidic droplets have a volume of less than about 10 nl).
Regarding claim 71, Weitz et al. teach hydrogel beads having a size in the range of 1-200 µm or in the range of 10-80 µm, or in the range of 20-70 µm (see pg 23, lines 5-13 and pg 88, claims 17-18: the limitation of an average diameter of at least about 1 µm and no more than about 500 µm).
Polansky (claim 76)
Polansky (US2004/0023207) taught (paragraph [0919]): “Well known advantages of commercial kits include convenience and reproducibility due to manufacturing standardization, quality control and validation procedures.”
It would have been prima facie obvious to an ordinary skilled artisan before the effective filing date of the instant invention, to apply the teachings of Zilionis et al., and Weitz et al. to the single cell barcoding method using droplet technology taught by Pellegrino et al. by utilizing the droplet volume guidelines and barcoded hydrogel bead particle diameter guidelines, microcapsule diameter taught by these one or more the cited references as suitable for barcoding using droplet microfluidics technology.
The ordinary skilled artisan would also have been motivated to optimize these parameters to enhance the performance of the method. It has been held that where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. In re Aller, 105 USPQ 233.
The ordinary skilled artisan would have been motivated convert RNA released from semi-permeable capsule into cDNA prior to its’ combination with the barcoded hydrogel bead for barcoding with a reasonable expectation of success as the cited art teach cDNA droplet barcoding as a matter of routine practice in the art. The ordinary skilled artisan would have been readily privy to use enzymes e.g. ExoI taught by Pellegrino et al to fragment and/or remove unwanted contaminants, to enhance downstream processes such as amplifying/barcode labeling.
Finally, would have been prima facie obvious to an ordinary skilled artisan before the effective filing date of the instant invention, to combine reagents suitable for single cell barcoding using droplet microfluidics as taught by Pellegrino et al., Zilionis et al., and Weitz et al. into a kit for practice of the method as Polansky teach benefits of kits e.g. convenience of having elements of practicing an assay or method in one place. In view of the combined teachings of all of the cited reference(s), the instant claims 51-76 are prima facie obvious.
Conclusion
No claims are currently allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to OLAYINKA A OYEYEMI whose telephone number is (571)270-5956. The examiner can normally be reached Monday -Thursday: 9:00 am - 5:00 pm, EST.
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OLAYINKA A. OYEYEMI
Examiner
Art Unit 1681
/OLAYINKA A OYEYEMI/Examiner, Art Unit 1681
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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