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
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Claim Objections
Claim 13 is objected to because of the following informality: line 3 states “…identical cell barde…” which should read as “barcode”. 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 4 and 37 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.
Claim 4 recites the limitation "…comprises sequencing the…amplification products thereof, or a portion of the…amplification products thereof." in lines 2 and 3. There is insufficient antecedent basis for this limitation in the claim as neither amplification products nor an amplification step had been previously recited.
Claim 37 recites the limitation "…the second covalent bond…" in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 102
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.
Claims 1, 26, 30, 34-38, and 45 are rejected under 35 U.S.C. 102(a)1 and (a)2 as being anticipated by Gehring [US 2019/0276818 A1].
Regarding claims 1, 26, 30, and 34-38, Gehring teaches a method for chemically labeling a plurality of samples comprised of one or more particles (such as cells) [Gehring, 0005, 0020].
For each sample, Gehring first incubates one or more amine-modified sample tags with a first heterobifunctional linker (aka linker) to generate one or more tetrazine-modified sample tags. The tetrazine-modified sample tag(s), a second linker, and an individual sample are then incubated together to generate tagged particles [Gehring, 0005].
The linkers of Gehring are equivalent to the coupling and indexing agent of the instant application. The first linker (e.g. the indexing label) is comprised of a tetrazine moiety (e.g. second reactive group) and an amine-reactive functional group (such as a N-hydroxysuccinimide (NHS) ester) which interacts with the one or more amine-modified sample tags (e.g. sample-specific-indexing sequence) [Gehring, 0005]. These sample tags can be single stranded and may comprise a capture sequence (such as a poly d(A) region), a PCR primer region, and a barcode sequence [Gehring, 0018].
The second linker (e.g. the coupling agent) is comprised of a tetrazine-reactive moiety (such as trans-cyclooctene (TCO)) (e.g. first reactive group) and an amine-reactive functional group (such as an NHS ester) (e.g. coupling group) [Gehring, 0005]. The linkers are joined in-situ via inverse-electron demand Diels Alder (IEDDA) chemistry and the amine-reactive functional group of the second linker chemoprecipitates the linker to the cell surface via nucleophilic attack by accessible cellular amines [Gehring, 0137]. Therefore, both the bond between the linkers and the bond connecting the second linker to the cell surface are considered to be covalent bonds.
Regarding claim 45, Gehring discloses a kit for chemically labeling a plurality of samples comprised of one or more amine-modified sample tags, a first linker, and a second linker (which are discussed in detail in the claim 1 rejection), as well as instructions for its use [0088].
Claims 2, 4, and 9-13 are rejected under 35 U.S.C. 102(a)1 and (a)2 as being anticipated by Gehring [US 2019/0276818 A1] as evidenced by 10X Genomics (10X Genomics. Chromium™ Single Cell 3’ Reagent Kits v2 User Guide. Rev B. 2017).
Regarding claims 2, 4, and 9-13, Gehring teaches a methods for chemically labeling a plurality of samples comprised of one or more particles (such as cells) which can then be sequenced as a pool and computationally demultiplexed [Gehring, 0005, 0020, 0072].
The process of chemically labeling a plurality of samples with amine-modified sample tag(s), a first linker, and a second linker is discussed in detail in the claim 1 rejection.
After performing chemical labeling, Gehring pooled the samples and subjected them to a modified version of the 10x Genomics v2 Single Cell 3` seq Reagent kit protocol [Gehring, 0109]. This protocol is capable of separately indexing each cell’s transcriptome by partitioning a sample of cells such that the majority of partitions contain no cells and the remaining largely contain a single cell (10X Genomics, page 2-3).
In each of these partitions, primers containing an Illumina read 1 sequencing primer, a 16bp 10x barcode, a 10bp Unique Molecular Identifier (UMI) and a poly(dT) primer sequence are incubated with the lysate of the single cell producing full-length cDNA from poly-adenylated mRNA (10X Genomics, page 2). As the indexing label includes a poly(dA) region, it would be transcribed into cDNA alongside the endogenous mRNA and thus similarly barcoded. This barcoded cDNA is then amplified by PCR. Finally, a library is constructed in which additional primer sequences and a sample index are added to the PCR product. The resulting library is depicted below. The result of following this protocol is the production of Illumina-ready sequencing libraries (10X Genomics, page 2-3). After sequencing, Gehring demultiplexed the samples by associating the results of the sequence analysis of the one or more particles with the sample of origin based on the sequence analysis of the one or more sample tags [0025].
PNG
media_image1.png
157
755
media_image1.png
Greyscale
Claim Rejections - 35 USC § 103
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.
Claims 27-29 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Gehring [US 2019/0276818 A1] in view of Wang (Wang M et al. Theranostics. 2016 Apr 12;6(6):887).
The limitations of claim 1, from which claims 27-29 and 33 ultimately depend, have been previously discussed and rejected in the 35 USC § 102 section.
Regarding claims 27-29 and 33, Gehring does not teach the inclusion of a hydrophilic group in the coupling and indexing agent.
However, Wang teaches that modifying TZ- and TCO- conjugated molecules with PEG spacers, in the context of biorthogonal reactions, enhance their solubility and thus improve their distribution in aqueous media (Wang, abstract). As the instant application is aimed at labeling cell membranes using a TZ/TCO system, a skilled artisan looking to improve the functionality of Gehring’s linkers in an aqueous (biological) environment would be motivated to include PEG spacers in order to maximize the number of cells effectively indexed.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Kara N Kovach whose telephone number is (571)272-8134. The examiner can normally be reached Monday - Friday, 9am - 3pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at (571) 272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681