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 .
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on Mar. 20, 2026. Claims 1, 3-5, 9-10, 12, 18, 20-22, 24-25, 28, 30-40 and 42-46 are pending. Claims 25, 31, 34 and 38-40, and 42-43 are withdrawn. Claims 1, 3-5, 9-10, 12, 18, 20-22, 24, 28, 30, 32-33, 35-37 and 44-46 are currently examined.
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.
(Previous rejection-maintained) Claims 1, 3-5, 9-10, 12, 18, 20-21, 24, 28, 30, 32-33 and 35-37 are rejected under 35 U.S.C. 103 as being unpatentable over Blainey et al. (WO 2016/149422 A1, published on Sep. 22, 2016) in view of Lee et al. (Wiley Interdiscip Rev Syst Biol Med. 2017 Mar;9(2):e1369, Epub 2016 Dec 21.).
The amended base claim 1 is directed to an in-situ method for screening cells for presence of one or more genetic elements comprising:
a) culturing one or more cells or a cell population in one or more discrete volumes;
b) introducing a plurality of polynucleotides into the cell or cell population by a lentiviral or retroviral system,
wherein each polynucleotide comprises one or more genetic elements,
wherein the genetic elements of the plurality of polynucleotides comprises a pool of combinatorial CRISPR-Cas based perturbations and a genome-wide library of sgRNAs, and
wherein each sgRNA is assigned an optical barcode such that each sgRNA comprises a different barcode, wherein each optical barcode is about 4 bp to about 32 bp in length;
c) incubating the cell or cell population to allow for expression of RNA transcripts comprising the one or more optical barcodes;
d) detecting genomic, genetic, epigenetic, proteomic, and/or phenotypic differences caused by the one or more genetic elements in the cell or cell population; and
e) generating a cDNA copy of the RNA transcripts, amplifying the generated cDNA copy by rolling circle amplification, and detecting the optical barcode by padlock in situ sequencing to identify the one or more genetic elements present in the cell or cell population.
Blainey et al. describes the encoding of DNA vector identity via iterative hybridization detection of a barcode transcript. They teach a new genetic perturbation and screening method that combines advantages of pooled perturbation with imaging assays for complex phenotypes. Specifically, the method may be used to screen pooled genomic perturbations to identify phenotypes and to identify perturbed genes at the single-cell level using optical barcodes. A major advantage offered by this approach is the ability to screen for any cellular phenotype that can be identified by high-resolution microscopy - including live-cell phenotypes, protein localization, or highly multiplexed expression profile and mRNA localization by RNA-FISH - in conjunction with a large array of genetic perturbations applied as a pool in a single test volume (See Abstract). Blainey et al. discloses that the embodiments disclosed herein provide in situ approaches based on serial probing with labeled oligonucleotides, and are highly suited for screening in cultured and primary cells (See [0020]). Therefore, Blainey et al. teaches the base claim 1 a) for culturing and screening cells for genetic modifications comprising culturing a cell or cell population in one or more discrete volumes (See [0003]), teaches the base claim 1 b) at introducing a plurality of polynucleotides into the cell or cell population by a lentiviral or retroviral system by stating that one or more vectors, such as a viral vector, are delivered into the individual cell or population of cells in each discrete volume. The vectors comprise nucleic acid sequences that encode one or more optical barcodes assigning to the sgRNA (See e.g., [0028]) and one or more genetic perturbations (See [0003]). Here the vector is lentiviral vector (See [0031]) and the library is sgRNA library for genome-wide screening (See [0001] and [0006]), and the genetic perturbations can be pooled CRISPR-Cas based genomic perturbations (See e.g., [0001] and claim 23). Blainey et al. teaches the base claim 1 c) and d) by stating that delivering a vector from the Cas9 library into the cell or cell population thereby introducing the one or more genetic perturbations and the barcode into the cell or cell population; incubating the cell or cell population to allow for expression of an RNA transcript comprising the barcode and the sequence of the optical barcode is then detected for the phenotype and genotype (See e.g., claims 23; [0003]; [0035]).
As for the limitation “each sgRNA is assigned an optical barcode such that each sgRNA comprises a different barcode, wherein each optical barcode is about 4 bp to about 32 bp in length” in the base claim 1 b), Blainey et al. teaches that in certain example embodiments, the optical barcode may further comprise a unique molecular identifier (UMI). The UMI is a short nucleotide sequence that can be used as an identifier for a specific optical barcode. For example, use of a UMI can allow sequencing of just the UMI to identify the optical barcode encoded in a given vector (See [0024]) and also teaches that all subsequent sequencing of this barcode pool requires sequencing just the 20 bp UMI and adjacent sequence of interest (e.g. sgRNA), rather than the full-length barcode (See [0028]), where the barcode length is 20 bp and teaches the range of 4 bp to 32 bp in length as claimed.
As for the newly amended base claim 1 e), Blainey et al. teaches in certain example embodiments, a cDNA copy of the expressed RNA transcript is generated and detection of the optical barcode is achieved by sequential binding to the cDNA copy of the RNA transcript (See [0046]), and the optical barcode may be detected directly using an in-situ sequencing method (See [0048]). However, Blainey et al. is silent on the “amplifying the generated cDNA copy by rolling circle amplification, and detecting the optical barcode by padlock in situ sequencing…”.
Lee et al. discloses a quantitative approach for investigating the spatial context of gene expression (See Title and abstract). Lee et la. teaches that a padlock probe and rolling circle amplification (RCA)-based approach can capture, amplify, and image the DNA with single nucleotide resolution in fixed cells and tissues that was later followed by the report of transcriptome-wide fluorescent in situ sequencing (FISSEQ) (See page 2, left column, paragraph 2). Figure 2 of Lee shows that the circular padlock probe is then amplified using rolling circle amplification (RCA) that increases the number of barcode-binding sites by 100-fold or more for robust imaging, and more specific than smFISH (single-molecule fluorescence in situ hybridization) (See page 3, left column and below), which can allow for sequencing of various barcode associated with cell lineage tracing, signaling pathways, promoter activities, and Cas9-targeted gene perturbations in situ (See page 9, left column, paragraph 2).
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It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to introduce the padlock probe and rolling circle amplification (RCA)-based method of Lee into Blainey’s invention. As described above, Lee et al. teaches that the padlock probe and rolling circle amplification (RCA)-based approach can increase the sensitivity, specificity of the FISSEQ and can be applied to Cas9-targeted gene perturbations in situ, one of skill in the art would have been motivated to do so to combine the teachings of Blainey and Lee to arrive at an invention as claimed. There would be a reasonable expectation of success to develop such an in-situ method for screening cells for presence of one or more genetic elements as claimed in the instant application.
Accordingly, Blainey et al. teaches an in-situ method for identify the one or more genetic elements present in the cell, wherein the method including culturing cells, a lentiviral or retroviral system of delivering polynucleotides into cells and expressing RNA transcription, where each polynucleotide comprises nucleic acid sequences encoding one or more optical barcodes with a sgRNA library and one or more genetic elements. Here the optical barcode can be a 20 bp unique molecular identifier (UMI), and the cDNA is amplified by rolling circle amplification, and detecting the optical barcode by padlock in situ sequencing
Regarding claim 3, it requires that introducing one or more polynucleotides into the cell or cell population comprises at least two polynucleotides. Blainey et al. teaches one or more vectors, such as a viral vector, are delivered into the individual cell or population of cells in each discrete volume. The vectors comprise nucleic acid sequences that encode one or more optical barcodes and one or more genetic perturbations. Each genetic perturbation to be introduced is assigned a unique optical barcode (See [0003]).
Regarding claim 4, it requires that in step a) the one or more cells or the cell population comprise the same genotype.
Blainey et al. teaches that they develop a method for genome-wide screening of genetic perturbations combined with imaging assays for complex phenotypes to identify relationships between genotypes and phenotypes (See [0001]) and the polynucleotides construct are introduced into a single cell or population of cells for the detection of phenotype and genotype using optical barcodes (See [0032]). It is noted that Blainey does not teach or suggest that the cells in the population are genetically different. Although Blainey et al. does not explicitly use the word of “ the same genotype” of cells, it would be obvious for one of ordinary skill in the art to select the same genotype cells based on the experimental optimization as needed.
Regarding claim 5, it requires comprising two or more discrete volumes in step a), each discrete volume comprising one or more cells or cell population. Blainey et al. teaches a method for screening cells for genetic modification comprises culturing a cell or cell population in one or more discrete volumes. One or more vectors, such as a viral vector, are delivered into the individual cell or population of cells in each discrete volume (See [0003]). Blainey et al. teaches one or more discrete volumes, where “more” can be considered to comprise two discrete volumes as claimed.
Regarding claim 9, it requires that the one or more genetic elements target genes in a pathway or intracellular network. Blainey et al. teaches that cellular localization signals are known in the art and can be selected based on a desired target location for localizing the transcript in the cell. In certain example embodiments, the localization signal is a cellular nucleus localization sequence. In one example embodiment, the nuclear localization signal is a 3' UTR stem loop, including stem loops from viral transcripts and the IncRNA MALATI (See [0024]). Blainey et al. further teaches that the vectors further encode a site-specific nuclease capable of introducing the genetic perturbation into a target sequence within a cell or population of cells (See [0027]). Blainey et al. discloses that the RNA transcript comprising the barcode, further comprises a cell localization signal localizing the RNA transcript comprising the barcode to a specific location within the cell, where the cell localization signal is a nucleus localization signal (See claims 11-12). Based on the description above, here the nucleus localization signal is the 3’-UTR step loop which is part of the intracellular network.
Regarding claim 10, it requires that the one or more genetic elements cause gene knock-down, gene knock-out, gene activation, gene insertion, insertion of a foreign sequence tag, or regulatory element deletion. Blainey et al. teaches that the library may contain a set of plasmids or other suitable delivery vectors with each delivery vector encoding one or more genetic perturbations. The genetic perturbations may include a gene knock-in, a gene-knock out, or one or more nucleotide insertions deletions, substitutions, or mutations [0021]).
Regarding claim 12 is directed to wherein step d) comprises determining a phenotypic difference by capturing a microscopic image or time series of microscopic images of the cell or cell population; and correlate the phenotypic difference to the identified one or more genetic elements. Blainey et al. teaches that the method comprising determining an observed phenotype for each cell or cell population by capturing a microscopic image of the cell or cell population; and correlating the observed phenotype to the identified genetic perturbation (See claim 2, page 16).
Regarding claims 18 and 20, they require that the RNA transcripts comprising the one or more optical barcodes further comprise a cell localization signal or other sequence ultimately localizing the RNA transcripts or premature termination. Blainey et al. teaches that in certain example embodiments, the nucleic acid encoding the optical barcode may further comprise a premature termination signal to prevent translation of the RNA transcript comprising the optical barcode. In certain example embodiments, the optical barcode may further comprise a localization signal to localize the expressed RNA transcript comprising the optical barcode to a particular cellular location (See [0024]).
Regarding claim 21, it requires each optical barcode is about 5 bp to about 20 bp in length. Blainey et al. teaches that UMI can be a DNA barcode and all subsequent sequencing of this barcode pool requires sequencing just the 20 bp UMI and adjacent sequence of interest (e.g. sgRNA), rather than the full-length barcode (See [0028]), which comprises the range of 4 bp to about 32.
Regarding claim 24, it requires the lentiviral or retroviral system has reduced recombination activity, or template switching activity, or multiple integration activity.
Blainey et al. teaches that the lentiviral vectors further encode a site-specific nuclease capable of introducing the genetic perturbation into a target sequence within a cell or population of cells, such as the site-specific nucleases ZFN and a CRISPR system comprising a dCas9 and sgRNA (See ;0027]), which can specifically cleave target DNA so to reduce the template switching activity of the vector. Blainey et al. also teaches that the library may contain a set of plasmids or other suitable delivery vectors with each delivery vector encoding one or more genetic perturbations. The genetic perturbations may include a gene knock-in, a gene-knock out, or one or more nucleotide insertions deletions, substitutions, or mutations (See [0021]), which can reduce/change the recombination activity of the vector.
Regarding claim 28, it requires the reduced recombination or template activity comprises reduced hairpin formation or dimerization through modification, knockdown or knockout of lentiviral or retroviral genomic RNA, or lentiviral or retroviral protein involved in dimerization. Blainey et al. teaches that a pooled library of transcriptional effectors for introducing one or more genetic perturbations is designed and cloned into a suitable vector. For example, the library may contain a set of plasmids or other suitable delivery vectors with each delivery vector encoding one or more genetic perturbations. The genetic perturbations may include a gene knock-in, a gene-knock out, or one or more nucleotide insertions deletions, substitutions, or mutations. The genetic perturbation may be generated using, for example, CRISPER/Cas9, RNAi (siRNA and shRNA), TALEN, Zn Finger enzymes, site directed mutagenesis, or other genetic engineering methods known in the art, or a combination (See [0021]). Although Blainey et al. does not specifically point out the hairpin or dimerization, it would be obvious for one of ordinary skill in the art to use the technique of Blainey such as CRISPER/Cas9 or RNAi to modify the hairpin or dimerization structure to reduce the recombination or template activity of the lentiviral vectors in order to minimize the risk of unintended genetic alterations in the host cell genome.
Regarding claim 30, it is directed to a lentiviral or retroviral system comprises the genetic element in the 3' LTR of the lentiviral genome. Blainey et al. teaches that optical barcodes are combinatorically assembled and cloned into an existing lentiviral CRISPR library. The constructs are sequenced to match sgRNAs to barcodes (See [0008]), and any suitable vector for delivering the constructs to a single cell or population of cells may be used. In certain example embodiments, the vector is a viral vector. In another example embodiments, the viral vector is a lentiviral vector (Se [0031]). Although Blainey et al. does not explicitly point out the cloning site of the genetic elements in the lentiviral genome, it is a routine technique to select cloning sites for DNA recombination. Nevertheless, Fig. 8 of Blainey shows that the genetic elements are cloned in the 3' LTR regions of the lentiviral genome (See Fig. 8 below). Blainey et al. teaches that in one example embodiment, the nuclear localization signal is a 3' UTR stem loop, including stem loops from viral transcripts and the IncRNA MALATI (See [0024]),
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Regarding claim 32, it requires that the individual discrete volume is a droplet generated on a microfluidic device. Blainey et al. teaches that In certain example embodiments, the discrete volume may be a culture chamber in an array of culture chambers defined on a microfluidic device, or droplet generated on a microfluidic device (See [0032]).
Regarding claim 33, it requires that the cell or cell population is contained within or isolated from a tissue sample. Blainey et al. teaches that the constructs are introduced into a single cell or population of cells. The cells may be cultured cells, primary cells, post-mitotic cells, such as neural cells, and tissue sections (See [0032]).
Regarding claims 35-37, they require that the mammalian subject is a human subject and the biopsy sample is a tumor sample. Blainey et al. teaches that the barcoding and imaging technique works regardless of cell type, thus a cell line of low culture maintenance such as the human melanoma derived A375 line will be used (See [0058]), where the A375 cell is a cell line exhibiting epithelial morphology that was isolated from the skin of a 54-year-old, female patient with malignant melanoma (See https://www.atcc.org/products/crl-1619). Also, Blainey et al. teaches that the cells may be cultured cells, primary cells, post-mitotic cells, such as neural cells, and tissue sections (See [0032]), where the tissue sections can be from a biopsy sample from a mammalian subject or a human subject as claimed.
(Previous rejection-maintained) Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Blainey et al. (WO 2016/149422 A1, published on Sep. 22, 2016) in view of Lee et al. (Wiley Interdiscip Rev Syst Biol Med. 2017 Mar;9(2):e1369, Epub 2016 Dec 21.) as applied to Claims 1, 3-5, 9-10, 12, 18, 20-21, 24, 28, 30, 32-33 and 35-37 above and in view of Kukita et al. (DNA Res. 2015 Aug;22(4):269-77).
Claim 22 requires each optical barcode is 12 bp.
Blainey et al. teaches that their method allows short-read DNA sequencing
(e.g. Illumina) of the sgRNA-barcode pair. Full length optical barcodes may range from l-2kb. For efficient sequencing, the optical barcodes are first sequenced to match the full barcode to a unique identifier of 20 "N" bases at the 5' end of the barcode. Although Blainey et al. does not specifically teach that the barcode tag is 12 bp, it is a common knowledge and routine technique in the art that the length of the barcode can be modified and determined based on the gene selection goals and related factors.
Nevertheless, Kukita et al. teaches that Barcode tags from mapped reads were analyzed in each target region. They designed a 12-bp barcode tag. Kukita et al. discloses that although they designed 12-bp barcode tags, they obtained tags that were not 12-bp in length due to insertion/deletion errors that were detected during sequencing. They discarded tags that were <9 bp. To recover the maximum number of reads, 11- and 13-bp tags that only differed from a 12-bp tag by the insertion or deletion of a single base were grouped with the corresponding 12-bp tag (See page 271, left column, paragraph 2). It discloses that the Reads with the same barcode sequences were grouped together, and the barcode tags were assigned into 2-read bins according to the number of reads per tag. Then, the proportion of 12-bp tags in each bin was calculated, and the value (proportion) for each bin was averaged using the 11 bins around that bin (See page 271, left column, paragraph 3). Kukita et al. also teaches that the fraction of 12-bp tags obtained exceeded 95% of the recovered fraction, and little improvement was observed when using more stringent thresholds (See page 271, right column, paragraph 3), which indicates that the 12 bp can work for the High-fidelity target sequencing. It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to learn the 12-bp barcode design method and benefit of Kukita and modify the barcode tag length of Blainey to design a 12-bp barcode tag as claimed in the instant application.
(Previous rejection-maintained) Claim 44 is rejected under 35 U.S.C. 103 as being unpatentable over Blainey et al. (WO 2016/149422 A1, published on Sep. 22, 2016) in view of Lee et al. (Wiley Interdiscip Rev Syst Biol Med. 2017 Mar;9(2):e1369, Epub 2016 Dec 21.) as applied to Claims 1, 3-5, 9-10, 12, 18, 20-21, 24, 28, 30, 32-33 and 35-37 above and in view of Molina Gil (Doctoral thesis (Ph.D), UCL (University College London).
Claim 44 is directed to a method wherein each barcode comprises no more than 4 consecutive repeating bases or comprises a GC content between 25% and 75%.
Blainey et al. teaches that Barcodes are designed by assembling 20-mer probes filtered for GC content, secondary structure, cross-hybridization, and off-target binding to transcriptome of target cells (See 0009]). However, it is silent on the percentage range of the GC contents.
Molina Gil studies the Lentiviral vector packaging cell line development using genome editing to target optimal loci discovered by high-throughput DNA barcoding, and teaches that although the GC content did not represent a major concern in this study, a range between 40-60% range would have been the advisable. A balanced GC range avoids the formation of secondary structures that hamper denaturation and annealing of oligonucleotides. In the current barcode design, a more balanced GC content could be achieved some of the fixed nucleotides within the barcode sequence or in the surrounding nucleotides by switching from W (A or T) to S (G or C) (See page 172, paragraph 2). Here the suggested GC content percentage (40-60%) of Molina Gil is in the range of the GC percentage as claimed (25% and 75%).
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to introduce the GC contents percentage of Molina Gil into Blainey’s invention to arrive at an invention as claimed. One of skill in the art would have been motivated to do so because the stability for the high GC contents (40-60%) provided. There would be a reasonable expectation of success to develop a method as claimed because Molina Gil teaches that the barcode is coupled with sgRNA delivered by lentiviral vector using CRISPR-Cas9 technology (See page 3, paragraph 2).
(New Rejection) Claim 45 is rejected under 35 U.S.C. 103 as being unpatentable over Blainey et al. (WO 2016/149422 A1, published on Sep. 22, 2016) in view of Lee et al. (Wiley Interdiscip Rev Syst Biol Med. 2017 Mar;9(2):e1369, Epub 2016 Dec 21.) as applied to Claims 1, 3-5, 9-10, 12, 18, 20-21, 24, 28, 30, 32-33 and 35-37 above and in view of Chen et al. (Nucleic Acids Res. 2018 Feb 28;46(4):e22).
Claim 45 requires step (e) comprises performing padlock extension and ligation of the cDNA transcript in an extension-ligation reaction mix comprising 1-l00nM dNTPs.
Blainey in view of Lee teaches performing padlock extension and ligation by combining T4 DNA ligation and rolling circle amplification (RCA) (See Figure 2). However, it is not explicitly pointing the reaction component such as dNTPs.
Chen studies the efficient in situ barcode sequencing using padlock probe-based BaristaSeq and teaches that the BaristaSeq results in a five-fold increase in amplification efficiency, with a sequencing accuracy of at least 97% (See Abstract). Chen teaches an in vitro gap-filling assays containing 20 µM dNTP (See page 2, left column). Although the dNTP is not in the range of the 1-l00nM dNTPs as claimed, Chen teaches that many factors, including probe concentration, dNTP concentration, and polymerase choice can affect the efficiency of the padlock probe application (See page 4, right column) and optimize the concentrations of dNTP in in situ barcode sequencing (See Figure 2 and below).
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In addition, according to section 2144.05 of the MPEP, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”). Since Chen teaches the importance of the dNTP concentration optimization, one of ordinary skills would be able to test for an optimal dNTPs concentration as claimed through routine experimentation unless there is evidence showing that they produce unexpected results.
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings from Blainey, Lee and Chen to arrive at an invention as claimed. One of skill in the art would have been motivated to do so because the concentration of dNTP is one of the key elements for affecting the extension-ligation reaction. There would be a reasonable expectation of success to develop the claimed method by performing padlock extension and ligation of the cDNA transcript in an extension-ligation reaction mix comprising the claimed dNTPs.
(New Rejection) Claim 46 is rejected under 35 U.S.C. 103 as being unpatentable over Blainey et al. (WO 2016/149422 A1, published on Sep. 22, 2016) in view of Lee et al. (Wiley Interdiscip Rev Syst Biol Med. 2017 Mar;9(2):e1369, Epub 2016 Dec 21.) as applied to Claims 1, 3-5, 9-10, 12, 18, 20-21, 24, 28, 30, 32-33 and 35-37 above and in view of Mohme et al. (Mol Ther. 2017 Mar 1;25(3):621-633) as evidenced by Bio-Rad (https://www.bio-rad-antibodies.com/starbright-dye-fixation.html).
Claim 46 is directed to method of claim 1, wherein the detecting the optical barcode of step (e) comprises fixing the one or more cells in a fixative comprising glutaraldehyde after the cDNA copy of the RNA transcripts is generated.
Blainey in view of Lee teaches a method for detecting the optical barcode and fix cells after the cDNA copy of the RNA transcripts is generated (See Blainey et al., e.g., [0004]; Lee et al., e.g., page 3), however, it is silent on using glutaraldehyde.
Mohme studies the optical barcoding for single-clone tracking to study tumor heterogeneity and teaches cells were fixed with 1% glutaraldehyde for 15 min and washed in PBS. (See page 631, right column, paragraph 1), and discloses that the fluorescent cell barcoding (FCB) represents another, flow cytometry-based labeling technique, which utilizes fluorescent dyes for staining of fixed cells resulting in a unique signature due to defined fluorescence intensities and emission wavelengths for different samples (See page 621, right column, paragraph 2). The advantage for using glutaraldehyde fixing cells can be evidenced by Bio-Rad’s teachings. Bio-Rad discloses that Glutaraldehyde fixes cells by cross-linking proteins via amino groups and is considered a stronger fixative than formaldehyde.
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings from Blainey, Lee and Mohme to arrive at an invention as claimed. One of skill in the art would have been motivated to do so based on the benefit by utilizing fluorescent dyes for staining of glutaraldehyde-fixed cells taught by Mohme. There would be a reasonable expectation of success to develop a method of fixing cell by glutaraldehyde as claimed.
Responses to Applicant’s Remarks
Applicant’s arguments filed on Mar. 20, 2026 has been received and fully considered.
Regarding the Rejections Under 35 U.S.C. § 103, Applicant’s argument is not found persuasive as follows:
1). Applicant argued that the person of ordinary skill would not have been motivated to combine the poorly scaling padlock-based approach of Nilsson described in Lee with Blainey to reach the present claims (See Remarks, bridging page 9-10).
The argument is not persuasive.
First, the instant claims do not cite limitation requiring a “scalability” as argued.
Second, Nilsson’s padlock in situ method is also used by the instant application (See instant specification, Example 8, [0418]). At the same time, Lee uses the same Nilsson’s method to conduct their study as well.
Third, the padlock probe was first invented by Nilsson and his colleagues in 1994, it is reasonable for Lee to use the Nilsson method to compare with their smFISH method. The importance is that Lee discloses the detailed method of Nilsson with pointing to its advantage for increasing the number of barcode-binding sites by 100-fold or more for robust imaging (See Figure 2), which makes Lee applicable to be used as a combination reference with Blainey.
2). Applicant argued that Kukita and Molina Gil cannot cure these deficiencies. Kukita is relied on merely to teach that barcode tags can be a certain length. Molina Gil is relied on merely to teach the sequence and GC content of barcode tags (See Remarks, page 10).
The argument is not persuasive.
Kukita and Molina Gil teaches a specific limitation for claim 22 and 44, respectively. It is applicable for Blainey in view of Kukita and Molina Gil to teach “optical barcode is 12 bp” in claim 22 and “GC content” in claim 44 as claimed, respectively.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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 extension fee 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 date of this final action.
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/RUIXUE WANG/ Examiner, Art Unit 1672
/THOMAS J. VISONE/ Supervisory Patent Examiner, Art Unit 1672
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