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 the following species in the reply filed on March 27, 2026 is acknowledged: polyT sequence (claim 151), rotating during both reagent dispensing and imaging (claims 160 and 161), and providing UV light (claim 164).
Information Disclosure Statement
The information disclosure statement filed December 15, 2023 fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. Non-patent literature #5 is identified on the information disclosure statement as being 29 pages in length, however, only 17 pages are present in the application file. It has been placed in the application file, but the information referred to therein has not been considered.
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.
Claim 148-167 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 148 states “wherein the first substrate or the second substrate or both are rotated during the sequencing in (a) or (d), respectively.” This language is unclear. The use of “respectively” implies that the first substrate is rotated during (a) and the second substrate is rotated during (d). The inclusion of “or both” would encompass a third option which is not accounted for by this usage. Therefore, a skilled artisan would be unable to ascertain the step at which each substrate is meant to be rotated.
Claims 149-167 are rejected by virtue of their dependency on claim 148.
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 148-163 and 165-167 are rejected under 35 U.S.C. 103 as being unpatentable over Frisen [US 10030261 B2] in view of Lucero [US 20210317524 A1] and Barbee [US 20190153520 A1].
Frisen discloses a method for the localized detection of a nucleic acid in a tissue sample. First, an array (e.g. substrate) is provided upon which multiple capture probes are directly or indirectly immobilized. Each of these capture probes comprise at least a positional domain (e.g., barcode) which indicates the location of the probe on the array and a capture domain. A tissue sample is placed on the array and the nucleic acids of the sample hybridize to the capture domain of the capture probes. Once captured, the probes are used as primers to generate new DNA molecules which contain both the sequence of the nucleic acid from the sample and the positional domain of the probe. These newly generated DNA molecules are then released from the substrate and sequenced, directly or indirectly, thus correlating each sample nucleic acid to a specific location within the sample [Frisen, C5 L49 – C6 L12]. Advantageously, Frisen states that arrays which are used in the art in any form of sequence analysis may be used as the basis of the arrays of their method [Frisen C30 L52-57].
Frisen does not teach (1) a first sequencing step to produce indexed data, (2) immobilizing the spatially tagged analytes, or derivatives thereof, on a second substrate upon which sequencing occurs, or (3) where the substrates are rotated during the sequencing steps.
Regarding (1), Frisen’s method combines the known synthesis location of each capture probe (and corresponding positional domain), sequencing data, and imaging data of the biological sample on the array to determine the spatial location of the biological analytes [Frisen, C54 L52-64].
Lucero discloses methods for resolving spatial arrays in which the features of an array comprise a capture probe immobilized to a surface, such as the surface of a bead [Lucero, abstract, 0096]. Each capture probe can include a spatial barcode, a constant sequence, and a capture domain. By determining the complete sequence of each capture probe, the spatial barcodes are thereby associated to a specific feature of the array [Lucero, 0009]. Once this is complete, biological analytes can be added to the array and captured by the capture domain of the probes present at each feature [Lucero, 0030].
Advantages of this method include that the barcodes can be sequenced through the use of high-throughput methods, including sequencing by synthesis methods using modified nucleotides (e.g., TruSeq™ and HeliScope™); and its ability to be adapted for use on existing arrays, including barcoded bead arrays which can be commercially manufactured to allow for millions of oligonucleotides to be attached to the array, thereby increasing the resolving capabilities of the system [Lucero, 0455, 0458, 0459, 0933].
Therefore, one of ordinary skill in the art prior to the effective filling date of the claimed invention, would be motivated to use the barcode sequencing method of Lucero while performing Frisen’s method in order to simplify array creation, increase throughput capabilities, and improve the overall resolution of the system. By utilizing commercial products and methodologies, cost-per-run of the method would likely be decreased as well. Simply put, the combination of familiar elements is likely to be obvious when it does no more than yield predictable results. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, A.).
Regarding (2) and (3), Barbee discloses a system for sequencing a nucleic acid molecule in which a substrate (e.g., planar array) is configured to both immobilize a biological analyte and rotate during sequencing [Barbee, 0062]. This substrate is comprised of individually addressable locations to which an analyte may be immobilized via a support, such as a bead [Barbee, 0177]. As Frisen explicitly states that any array capable of sequencing may perform the second sequencing step, a skilled artisan would be motivated to utilize any sequencing chemistry available to them. As the array disclosed by Barbee was known in the art prior to the effective filing date of the claimed invention, and is capable of performing the task required by Frisen, the skilled artisan would have recognized Barbee’s array as an option available to them.
Therefore, by modifying the method of Frisen to include the barcode sequencing steps of Lucero and performing said modified method on the array of Barbee, a skilled artisan would arrive at the claimed invention in which indexed data and sequencing data are combined to provide spatial data of an analyte.
Regarding claim 149, Lucero states that the substrate (e.g. a bead) can include tens to millions of individual oligonucleotide molecules and may be considered the “capture probe” containing a spatial barcode [Lucero, 0263, 0386]. The spatial barcode by be common to all of the capture probes attached at a given feature (i.e., bead) and which is unique relative to the spatial barcodes of capture probes associated with other features [Lucero, 0009, 0265].
Regarding claims 150, 151, and 153, Frisen states that the capture domain of the capture probe comprises a nucleotide sequence that is capable of hybridizing to a nucleic acid from the tissue sample. In the preferred embodiment, the capture sequence comprises a poly-T DNA oligonucleotide for use in capturing mRNA molecules through hybridization to their poly-A tail [Frisen, C13 L27-45].
Regarding claim 154, Frisen states that an array may contain 2-4,200,000 individual features (i.e., individually addressable locations) [Frisen, C20 L10-24].
Regarding claim 155, Barbee states that the array may be coated with binders on at least 99% of the individually addressable locations. These binders can immobilize the analytes through non-specific interactions, including electrostatic interactions. Barbee also states that the analytes bound at the individually addressable locations may include the beads [Barbee, 0184-0190]
Regarding claims 156 and 157, after the completion of Frisen’s method and per the previously outlined rationale, the resulting spatially-tagged DNA molecules, would be introduced to a second array of Barbee and subsequently immobilized to an individually addressable location on Barbee’s array via a bead [Barbee, 0062, 0111, 0177]. Once bound, chemical reactions may be performed prior to sequencing, such as nucleic acid amplification, to form a colony of a derivative of the spatially tagged analyte on the bead which would increase the signal detected from the analyte [Barbee, 0192].
Regarding claims 158 and 159, and as outlined above, sequencing by synthesis methods can be used to analyze the barcode sequences in step (a) [Lucero, 0781]. When performing sequencing by synthesis, a plurality of nucleotides are provided as single bases which may be the same type of nucleotide and may be a mixture of labeled and unlabeled nucleotides [Barbee, 0207, 0209]. Multiple iterations of this may be performed such that each provision of nucleotide solution is distinct from one another [Barbee, 0216].
Regarding claims 160 and 161, Barbee states that reagents may be dispensed onto the substrate prior to or during rotation and that detecting can be conducted using a sensor that continuously scans the array during rotation of the substrate [Barbee, 0177].
Regarding claim 162, the arrays of Barbee are substantially planar [Barbee, 0010].
Regarding claim 165 and 166, Frisen presents Example 10 in which a formalin-fixed frozen tissue was permeabilized prior to performance of the method [Frisen, C72 L14-26].
Claim 164 is rejected under 35 U.S.C. 103 as being unpatentable over Frisen, Lucero, and Barbe as applied to claims 148 and 163 above, and further in view of Bian [Bian Q et al. ACS applied materials & interfaces. 2016 Oct 12;8(40):27360-7] and Royes [Royes J et al. The Journal of Organic Chemistry. 2020 Apr 17;85(10):6509-18].
Regarding claim 164, Frisen states that the releasing step may be achieved using a number of methods which may or may not include DNA cleavage (Frisen, C31 L710]; Lucero states that the capture probe may be linked to a feature via a UV sensitive bond [Lucero, 0746]; and Barbee states that the binder may be cleavable [Barbee, 0209, 0225]. Therefore, while all three teach that the inclusion of a cleavable bond is possible, with Lucero specifically stating that this bond may be UV cleavable, none teach the specifics of the UV sensitive bond as claimed by the Applicant (i.e., oligonucleotide molecules conjugated to azobenzene which is bound to an alpha-cyclodextrine moiety beads).
However, Bian describes the capture and light-triggered release of specific cancer cells using a β-cyclodextrin (β-CD)/azobenzene (AZO) system. This system is generated in three steps: (1) a silicon substrate is modified to include β-CD molecules; (2) thio-AZO is attached to the β-CD molecules; and (3) a specific cell capture agent, a target-specific oligonucleotide modified with an amino linker, is connected to the thio-AZO. When constructed, this system will capture specific cells and can be made to subsequently release them through UV light irradiation. This exposure induces a trans- to cis- confirmation change in AZO which causes it to disassociate from β-CD, thus releasing the (β-CD)-oligo-captured cell complex. Additionally exposing the system to visible light reverses azobenzene’s confirmation change allowing it to associate with β-CD once again, thus making this a reusable system (Bian, p27360-61, Fig. 1A).
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One benefit of this system is the fact that oligonucleotides are easy to synthesize and are highly specific for a chosen target molecule increasing the diversity of targets this system is capable of capturing. A second benefit is this system is reuseable. Multiple capture cycles can be performed by reattachment of the (β-CD)-oligo complex. A skilled artisan would recognize the experimental flexibility these two benefits provide. The same azo-modified substrate could be used to capture multiple different types of targets by simply changing the oligonucleotide in the (β-CD)-oligo complex. Finally, Bian teaches that the use of planar substrates results in low cell-capture efficiency, and suggests the use of microstructures to increase the contact area, and thus adhesion efficiency, between the capture system and the target cells. (Bian, p27360, 64)
Therefore, one of ordinary skill in the art prior to the effective filling date of the claimed invention, looking for an effective UV light-based cleavage mechanism for use in the method of Frisen, Lucero, and Barbee, would have been motivated to apply the teachings of Bian and incorporate a CD/AZO system. Since the specificity of Bian’s system is dependent on the oligonucleotide used, and oligonucleotides can be used to capture complementary nucleic acids, the skilled artisan would recognize its applicability to spatial screening of analytes. As Bian teaches that microstructures are more effective for capture than planar substrates, the inclusion of this cleavage chemistry on beads would be obvious. Additionally, the reusable nature of this system facilitates the cleavage of the oligonucleotides from the beads and their subsequent re-immobilization to a second substrate. The combination of familiar elements is likely to be obvious when it does no more than yield predictable results. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, A.).
Bian, however, teaches the use of β-CD in the CD/AZO system, whereas the Applicant claims the use of α-CD.
Royes modelled the thermodynamic and photokinetic parameters of azo/CD complexes in aqueous solutions and compared how the use of α-, β-, and γ-CD impacts those characteristics (Royes, p6510). As a result, Royes reached the following pertinent conclusions:
α-CD is able to establish more effective interactions with trans-AZO than β- or γ- CD (Royes, p6510 C2).
β-CD is capable of complexing with cis-AZO, whereas α-/γ-CD are not (Royes, p6511 C1).
Both α- and β- CD prefer 1:1 complexing with AZO (Royes, p6511 C2).
α-CD complexes are more stable than β-CD complexes (Royes, p6513 C1).
Royes points out that previous studies were performed in organic solvents, or their aqueous mixtures. Therefore, the assertion that β-CD exhibits a higher affinity for trans-Azo as opposed to cis-AZO when applied to biological systems is likely fallacious as Royes’ results revealed equal affinities in aqueous systems (Royes, p6510 C1, p6513 C2).
Therefore, one of ordinary skill in the art prior to the effective filling date of the claimed invention, would have been motivated to use alpha-CD instead of beta-CD in the method of Frisen, Lucero, Barbee, and Bian as Royes teaches that alpha-CD performs better in the aqueous environments in which their method is intended to be used. Additionally, as beta-CD can remain complexed with cis-AZO, its entirely likely that a portion of the oligonucleotides would not be released and thus would not participate in the subsequent steps of the method.
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.
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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