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 .
Abstract
Applicants correction of the Abstract is noted and the objection is withdrawn.
Specification
Applicants correction of the informalities in the Specification is noted and the objection is withdrawn.
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.
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 1-9 are rejected under 35 U.S.C. 103 as being unpatentable over Frisen (US PG Pub
2020/0399687, US Patent Doc 1 on IDS dated 09/08/2022) in view of Cai (WO 2018026873,
published 02/08/2018) and Haller et al (2016).
Regarding claim 1, Frisen teaches a method of spatially tagging an mRNA sample from a tissue
sample (e.g., Frisen para 0150), and sequencing the cDNA produced from the tagged mRNA (e.g., Frisen
para 0153) using a flow cell and fiducial marker (e.g., Frisen paras 0150, 0126) reading on the limitations
“A method to obtain the spatial location and sequence information of an m-RNA target sequence on a
tissue sample” and “providing a solid surface having at least with at least one fiducial marker” of the
instantly rejected claim 1 steps a. Frisen teaches binding a first molecule, reading on the anchor molecule, and a second molecule, which has a barcode and polyA tail, reading on the scaffold molecule, to the first molecule and extending it to create a complementary barcode and polyT tail, then removing the second molecule (e.g., Frisen Fig 1A), and where the barcode is 27mers and randomly generated during synthesis, and the polyT tail is 24mers (e.g., Frisen paras 0151 and 0154) reading on the limitations “attaching a plurality of anchor molecules comprising a” “cleavable linker and an adapter unit to the solid surface “, “Binding scaffolding molecules comprising a unit capable of binding to the adapter unit of an anchor molecule, a poly-inosine unit having 5 to 30 inosine bases and a poly-adenine unit having 10 to 50 adenine bases to the anchor molecules”, “incorporating thymine to the anchor molecules complementing the poly-adenine unit of the scaffolding molecules thereby creating a poly-T unit”, “removing the scaffolding molecules from the anchor molecules” of the instantly rejected claim 1 steps b, c, e and f.
Frisen teaches providing a tissue sample with mRNA, capturing the mRNA with the polyT tail (e.g., Frisen para 0160, 0153, Fig 1B) and reverse transcribing the mRNA creating a cDNA strand attached to
the surface, then cleaving and removing it from the surface (e.g., Frisen Fig 1B) reading on the limitations “providing a tissue sample comprising at least one m-RNA strand wherein at least one m-RNA strand of the sample binds to a poly-T unit of at least one anchor molecule”, “reverse transcription of the m-RNA strand creating a c-DNA strand attached to the solid surface” and “remove c-DNA strands from the solid surface” of the instantly rejected claim 1, steps g, h and i. Frisen teaches preserving the spatial information of nucleic acids, while performing sequencing on the cDNA strands and detecting the fiducial markers in the same image (e.g., Frisen para 0024; Fig 1; para 0127) reading on the limitations “obtaining the sequence information of the c-DNA strands and linking the spatial information with the sequence information of the c-DNA strands” and “herein the sequence of the barcodes and their spatial location relative to the fiducial marker is detected simultaneously as spatial information” of the instantly rejected claim 1, steps d and j.
Although Frisen does teach cleavable linkers attached to a solid surface, they do not particularly
teach photo-cleavable linkers nor do they teach the use of Inosine bases for randomly incorporating
nucleotides into amplification products, but these were known in the art and were taught by Cai and
Haller et al.
Cai teaches a method of detecting mRNA in tissue (e.g., Cai example 4, para 00407), where the
binding sequence is connected via a photo-cleavable linker (e.g., Cai para 0040, 0067).
It would have been prima facie obvious before the effective filing date of the claimed invention
for a person having ordinary skill in the art to have used a photo-cleavable linker of the method of Cai for obtaining spatial and sequence information from mRNA in tissue with the method for obtaining spatial and sequence information from RNA in a tissue sample of Frisen. The skilled artisan would have been motivated to use the photo-cleavable linker by the teachings of Frisen that the cleavage site may be broken by specific processes, such as chemical, enzymatic or physical (e.g., Frisen para 0035), and by the teachings of Cai that their invention provides technologies that are useful for profiling transcripts or DNA loci in cells, tissues or organs and overcomes problems associated with prior methods of (e.g., Cai paras 0005, 0007). Thus, the addition of the photo-cleavable linker of the method of Cai to the method of Frisen would have been a simple combination of known methods to yield predicable results.
Haller et al teaches a method of targeted insertion of Inosine into template nucleic acid to
introduce randomized nucleotides (i.e., A, G, C, T) to the amplified product of the template strand (e.g.,
Haller et al., page 923, left column, para 2; online methods, page 925, left column, para 2) reading on the limitation “randomly incorporating adenine, guanine, cytosine and thymine as nucleic bases to the anchor molecule complementing the inosine bases of the scaffolding molecules thereby creating barcodes” of the instantly rejected claim 1, step d.
It would have been prima facie obvious before the effective filing date of the claimed invention
for a person having ordinary skill in the art to have used the random incorporation of nucleotides to
amplified product of Haller et al with the method for obtaining spatial and sequence information from
RNA in a tissue sample with random barcodes of Frisen. The skilled artisan would have been motivated
to use the random incorporation of nucleotides of Haller et al by the teachings of Haller et al, that Inosine has the innate characteristic of base-pairing with each of the four nucleotide bases (e.g., Haller et al., page 923, left column, para 2) and that nucleotide bases are randomly incorporated in place of inosine during amplification (e.g., Haller et al., online methods, page 925, left column, para 2), and the skilled artisan desiring to create random barcodes, would understand the utility of inosine for this incorporation. Thus, the addition of the random incorporation of nucleotides of Haller et al to the method of Frisen would have been a simple combination of known methods to yield predicable results.
Regarding claim 2, Haller et al teaches a method of random incorporation of adenine, guanine,
cytosine and thymine as nucleic bases to the first molecule by providing dNTPs and Firebird Taq 475
(i.e., polymerase) (e.g., Haller et al., online methods, page 925, left column, para 2).
Regarding claim 3, Frisen teaches a method of obtaining spatial information from fluorescently
detectable nucleotides via sequencing-by-synthesis on commercially available platform (i.e., Illumina®
platforms) (e.g., Frisen para 0083, 0123, 0154-01544).
Regarding claim 4, Frisen teaches a method of obtaining spatial information from fluorescently
detectable nucleotides via sequencing-by-synthesis on commercially available platform (i.e., Illumina®
platforms) (e.g., Frisen para 0083, 0123, 0154-01544).
Regarding claim 5, Frisen teaches a method where the sample tissue is removed from the surface (e.g., Frisen para 0160).
Regarding claim 6, Frisen teaches a method of amplifying the target cDNA via rolling circle
amplification (e.g., Frisen 0076, 0077).
Regarding claim 7, Frisen teaches a method wherein the nucleic acid probe attached to the solid
support, reading on the anchor molecule, is randomly distributed on the solid support (e.g., Frisen para
0006), with density controlled by the concentration of anchor molecules loaded on the substrate and wherein the molecules have a minimal distance from each other of between about 50-300nm (e.g., Frisen para 0040).
Regarding claim 8, Frisen teaches a method wherein the tissue sample can be attached to a solid
support and can be permeabilized (e.g., Frisen para 0097).
Regarding claim 9, Frisen teaches a method where the first molecule, reading on the anchor
molecule, comprises an SBS primer, which may be extended via rolling circle amplification (e.g., Frisen
Fig 1, 0076, 0077).
Response to Arguments
Applicant’s arguments with respect to the objections over claims 1 and 6, filed November 11, 2025, have been fully considered and are persuasive. The objections have been withdrawn.
Applicant’s arguments with respect to the 112b rejections over claim 7, filed November 11, 2025, have been fully considered and are persuasive. The rejections have been withdrawn.
With respect to the 103 rejection, applicant argues that prior-art arrays rely on pre-defined barcodes and that the claimed random in situ SBS synthesis and concomitant decoding is a fundamental change in principle and teaches away from in situ barcode generation.
The Office acknowledges that Frisen and Cai emphasize pre-fabricated barcode approaches. However, the examiner finds that teaching away requires a clear suggestion in the prior art to avoid the claimed approach or a clear discouragement of the modification. The cited passages in Frisen and Cai describing pre-fabricated arrays do not explicitly or unequivocally teach away from on-surface synthesis or randomized in situ barcode generation; they describe a particular implementation (pre-fabricated arrays) but do not state that in situ generation is infeasible or undesirable in all embodiments.
The fact that prior-art implementations used pre-defined barcodes does not alone mean the prior art teaches away from on-surface barcode synthesis; it merely shows an alternative approach.
Haller’s concerns about the challenges of single-molecule decoding are acknowledged, but reporting challenges or stating that a problem exists is not equivalent to teaching away from attempting an alternate solution. Haller’s disclosure of SBS imaging and fiducial-based registration provides technical teachings that would support attempting an alternative approach and does not constitute a teaching away.
Additionally, applicant argues that mixed-nucleotide incorporation opposite inosine would suffer polymerase bias/stalling and high error rates, rendering the approach unsuitable. This is an evidence-based technical assertion. However, Applicant has not submitted experimental evidence (e.g., declarations, data, or comparative examples) in the record demonstrating that the approach would be inoperable, that polymerase bias cannot be overcome, or that the claimed combination would not work. Absent such evidence, it is presumed that a skilled artisan could reasonably optimize polymerase, buffer, and reaction conditions to perform SBS on a suitable template (including universal bases or suitable oligonucleotide designs) to generate informative barcodes. Further the presence of potential biases or inefficiencies in polymerase behavior opposite inosine does not establish lack of reasonable expectation of success if the references or knowledge in the field provide methods to mitigate such issues (e.g., selection of appropriate polymerases, buffer conditions, modified nucleotides, cycle parameters) as discussed in the teachings in the rejection above.
Applicant argues use of hindsight reasoning. This argument is not persuasive. A proper obviousness analysis may consider whether the combined teachings would have provided an ordinary artisan with some reason to attempt the combination with a reasonable expectation of success. Here, Haller’s SBS and fiducial registration teach high-resolution sequence/position registration while Frisen and Cai teach surface immobilization and capture chemistry. These teachings provide an objective basis for combining the methods to achieve spatially linked sequence information.
Summary
No claims allowed
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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HEATHER . CALAMITA
Supervisory Patent Examiner
Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684