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 .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 103-116, in the reply filed on 04/30/2026 is acknowledged.
Claims 117-122 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 04/30/2026.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 103-109 and 111-116 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gehring (2018) in view of Vigneault (WO 2017/053905, IDS ref).
Regarding claims 103-104 and 109, Gehring disclosed a method called SUGAR-seq (section 2.4, pages 24-26), in which samples of cultured cells or single cell suspensions are exposed to nucleotide sugars and glycan-specific transferases, which incorporate the modified sugars into cellular glycans (“First, a small, bioorthoganol functional group is installed on a sugar of interest. Then, the modified sugar is ligated to uridine diphosphate, forming the modified UDP-sugar. Finally, a glycosyltransferase is engineered to accept this modified sugar and install it onto protein substrates.”). See Fig. 2.8, page 25.
Next, sugar-specific barcodes are ligated to these incorporated modified sugars; Fig. 2.8 illustrates the case where the modified, incorporated sugar bears an azide group (N3) and is ligated to DBCO on the oligonucleotide barcode.
Next, individual cells are partitioned into droplets, each droplet containing a single cell and a microparticle conjugated to a unique oligonucleotide (serving as a partition or droplet-specific barcode), and, following cell lysis, a hybridization and reverse transcription step creates a nucleic acid comprising both the sugar-specific barcode sequence and the droplet-specific barcode sequence. See Fig. 2.8, page 25.
These nucleic acids are PCR-amplified and sequenced to determine glycan counts for each cell (Fig. 2.8 legend).
Regarding claims 105-107 and 113-115, Gehring disclosed performing “three sequential rounds of chemoenzymatic labeling to affix a unique, sugar-specific DNA-oligo barcode to each glycan” (Fig. 2.8 legend).
Regarding claim 108, Gehring taught the sample could be tissue culture cells, or individual cells obtained from tissue (Fig. 2.8 legend).
Regarding claim 111, in Gehring’s example, an azide group was used (Fig. 2.8), along with DBCO, which is capable of coupling thereto.
Regarding claims 112 and 116, Gehring disclosed (page 25, first paragraph under Fig. 2.8 legend): “…the bacterial homolog of the blood human blood group A antigen glycosyltransferase (BgtA) has been employed to transfer a GalNAz sugar to the C-3 position of galactose in fucose-α(1-2)galactose…”.
Gehring did not disclose “removing unincorporated reporter molecules from the sample” as recited in claim 103, step (c). Also, while Gehring disclosed “three sequential rounds of chemoenzymatic labeling to affix a unique, sugar-specific DNA-oligo barcode to each glycan”, suggesting first, second and third flag molecules/nucleotide molecules/reaction pairs/glycan-specific transferases/reporter oligonucleotides, Gehring does not explicitly suggest a fourth. In this regard, however, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the instant application to extend the method of Gehring to probe additional types of glycans using additional tranferases, orthogonal reaction pairs and reporter oligonucleotides in order to assess other types of glycans.
Vigneault taught a similar technique in which cell surface molecules were reacted with affinity-barcode conjugates, the cells were encapsulated in droplets containing “droplet barcodes”, and composite nucleic acid molecules containing both the affinity barcode and the droplet were produced; see Fig. 9A-C and paragraphs [0093]-[0098].
Vigneault taught washing the cells following the contacting of the cells with the conjugates in order to remove the unbound conjugates (paragraphs [00225], page 45).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the instant application to modify the method of Gehring by “removing unincorporated reporter molecules from the sample”, one would have been motivated to do this prior to partitioning individual cells. Otherwise, such unincorporated reporters would have been present in the partitions, where they would have also contributed to formation of nucleic acids containing the corresponding sugar-specific barcodes, and thereby have resulted in inaccurately high counts for the respective glycans for a given cell.
Claim(s) 110 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gehring (2018) in view of Vigneault (WO 2017/053905, IDS ref) as applied to claims 103-109 and 111-116 above, and further in view of Chen (WO 2019/126466).
The disclosures of Gehring and Vigneault have been discussed.
These references did not disclose or suggest encapsulating single cell lysates into a “cell bead”.
Chen disclosed a method wherein single cells and probes were encapsulated in droplets containing a polymerizable or gellable polymer, the cell lysed, and the polymer polymerized or gelled (thus forming a “cell bead”). The target of the probe is captured by the probe and subsequently barcoded; Fig. 1, paragraph [00203], page 75.
Chen points out advantages of this approach (paragraph [00107], page 41): “Firstly, they are dispersible in aqueous phase. This can be an important aspect because other water-soluble reagents such as enzymes, primers, barcode polynucleotides can diffuse into the hardened particle from the surrounding aqueous solution, or out of the hardened particle into the surrounding aqueous solution. This property distinguishes hardened particles from the water-in- oil droplets which are not dispersible in aqueous phase (i.e., they are only dispersible in oil phase). Secondly, they are non-mergable. This can be another important aspect because one needs to minimize the targets from different hardened particles from mixing. This property distinguishes hardened particles from droplets of highly viscous liquid (e.g., glycerol) which may merge with each other in routine laboratory handling. Because of the advantages, many steps can be performed after polymerizing or gelling of the polymers and/or monomers. For example, lysing entrapped cells, barcoding, washing, or primer extension (e.g. reverse transcription) can be performed after obtaining hardened particles.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method suggested by the combined teachings of Gehring and Vigneault by encapsulating the cells in a gel or polymer bead prior to lysis and barcoding because of the advantages pointed out for this approach by Chen.
Conclusion
No claims are free of the prior art.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SAMUEL C WOOLWINE whose telephone number is (571)272-1144. The examiner can normally be reached 9am-5:30pm.
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/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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