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 .
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 14-20 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 pre-AIA the applicant regards as the invention.
Since term “cBC” is not defined within claims 14, 19, 20 and no standard meaning in the art has been established for it in this context. As written, one of ordinary skill in the art cannot determine with reasonable certainty what structural or functional limitation is intended by “cBC”. Hence, the metes and bounds of the claim are unascertainable.
Claims 15-18 depend from claim 14 and are therefore similarly rejected.
As per MPEP 2173: It is of utmost importance that patents issue with definite claims that clearly and precisely inform persons skilled in the art of the boundaries of protected subject matter. Therefore, claims that do not meet this standard must be rejected under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph as indefinite. Further, as per MPEP 2173.02: If the language of the claim is such that a person of ordinary skill in the art could not interpret the metes and bounds of the claim so as to understand how to avoid infringement, a rejection of the claim under 35 U.S.C. 112, second paragraph, would be appropriate. As currently written, the metes and bounds of the rejected claims are unascertainable for the reasons set forth above, thus the above claim(s) and all dependent claims are rejected under 35 USC 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Liu et al.
Claim(s) 1 and 2 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Liu et al. (mSystems, 2018, Vol. 3, Issue 1).
Regarding claim 1, Liu discloses a plurality of expression vectors, wherein each expression vector of the plurality comprises a first identifying nucleic acid barcode (rBC) uniquely associated with the individual expression vector. (e.g. they construct a “magic pool” in which each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence [Fig. 1], [abstract]).
Regarding claim 2, Liu discloses the plurality of expression vectors of claim 1 as discussed fully above and incorporated here. Liu further discloses wherein the first identifying nucleic acid barcode (rBC) is a randomized sequence. (e.g. they construct a “magic pool” in which each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence [Fig. 1], [abstract]).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Liu et al. and Klein et al.
Claim(s) 3-13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (mSystems, 2018, Vol. 3, Issue 1) in view of Klein et al. (Nature Methods, 2020, Vol 17, pages 1083–1091).
Regarding claim 3, Liu discloses the plurality of expression vectors of claim 2 as discussed fully above and incorporated here. Liu further discloses each expression vector further comprises: a. a nucleic acid regulatory element; b. an open reading frame optionally encoding a reporter gene (e.g. the regulatory region that locates upstream of open reading frame including both the promoter and the ribosome binding site [Fig.1 and page 3]). However, Liu does not disclose a second identifying nucleic acid barcode (cBC) uniquely associated with the nucleic acid regulatory element; wherein the nucleic acid regulatory element of each expression vector is selected from a plurality of different nucleic acid regulatory elements.
Klein discloses identifying nucleic acid barcode (cBC) uniquely associated with the nucleic acid regulatory element; wherein the nucleic acid regulatory element of each expression vector is selected from a plurality of different nucleic acid regulatory elements. (e.g. a massive parallel reporter assay, in which thousands of regulatory elements, such as enhancers, and their variants are tested to elucidate effects of regulatory sequence variance on regulatory activities [abstract]. Klein further teaches building a library of enhancers in which each member is genetically unique. These sequences then introduced to cells using either episomal or integrated genetic material vectors, wherein the construction of vectors comprising regulatory elements locates upstream of a promoter and the enhancer associated barcodes is in the 3′ UTR of the reporter gene [Fig. 1 and page 1083-1084]).
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the plasmid library of Liu to include the regulatory element variant constructs taught by Klein to broaden the functional scope of pooled expression assays to include testing of diverse enhancer or promoter elements within same barcoded system. As of the application’ s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to combine these teachings to enable simultaneous identification of both the expression vector (rBC) and the specific regulatory element (rBC) responsible for transcriptional activity, since both Liu and Klein employ barcoded library for quantifying construct expression. Hence, the proposed combination constitutes a predictable use of prior-art elements according to their established functions and would have been obvious to one of ordinary skill in the art at the time of filing. This reasoning is consistent with KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143,A)
Regarding claim 4, Liu and Klein disclose the plurality of expression vectors of claim 3 as discussed fully above and incorporated here. Klein further discloses each nucleic acid regulatory element is a genetic variant of a single nucleic acid regulatory element. (e.g. building a library of enhancers in which each member is genetically unique. Klein teaches that each regulatory element or enhancer sequence represents a unique variant differing by one or more nucleotide substitution, deletion, or insertion, enabling systematic evaluation of regulatory sequence-function relationship. The construction of vectors comprising one regulatory element variant each, wherein the regulatory element locates upstream of a promoter and the regulatory element associated barcodes is in the 3′ UTR of the reporter gene [Fig. 1 and page 1083-1084]). Therefore, Klein teaches the additional limitation of claim 4.
Regarding claim 5, Liu and Klein disclose the plurality of expression vectors of claim 4 as discussed fully above and incorporated here. Klein further discloses each nucleic acid regulatory element differs from the remaining nucleic acid regulatory elements by at least one nucleotide substitution, deletion, or insertion. (e.g. building a library of enhancers in which each member is genetically unique. Klein teaches that each regulatory element or enhancer sequence represents a unique variant differing by one or more nucleotide substitution, deletion, or insertion, enabling systematic evaluation of regulatory sequence-function relationship. The construction of vectors comprising one regulatory element variant each, wherein the regulatory element locates upstream of a promoter and the regulatory element associated barcodes is in the 3′ UTR of the reporter gene [Fig. 1 and page 1083-1084]). Therefore, Klein teaches the additional limitation of claim 5.
Regarding claim 6, Liu and Klein disclose the plurality of expression vectors of claim 5 as discussed fully above and incorporated here. Klein further discloses the cis- regulatory element is an enhancer, promoter, insulator, or silencer. (e.g. massive parallel reporter assay in which thousands of cis-regulatory elements such as enhancers, promoters, and their variants, are tested to elucidate effects of regulatory sequence variation on transcriptional activity [abstract]. Klein teaches that each vector contains a regulatory sequence driving expression of reporter gene [Fig 1]). Therefore, Klein teaches the additional limitation of claim 6.
Regarding claim 7, Liu and Klein disclose the plurality of expression vectors of claim 6 as discussed fully above and incorporated here. Klein further discloses the cis- regulatory element is an enhancer, promoter, insulator, or silencer. (e.g. massive parallel reporter assay in which thousands of cis-regulatory elements such as enhancers, promoters, and their variants, are tested to elucidate effects of regulatory sequence variation on transcriptional activity [abstract]. Klein teaches that each vector contains a regulatory sequence driving expression of reporter gene [Fig 1]). Therefore, Klein teaches the additional limitation of claim 7.
Regarding claim 8, Liu and Klein disclose the plurality of expression vectors of claim 7 as discussed fully above and incorporated here. Klein further discloses cis- regulatory element is a core promoter. (e.g. massive parallel reporter assay in which thousands of cis-regulatory elements such as enhancers, core promoters (“minimal promoter”), and their variants, are tested to elucidate effects of regulatory sequence variation on transcriptional activity [Fig.1, method section “Library cloning”]. Klein teaches that each vector contains a regulatory sequence driving expression of reporter gene [Fig 1]). Therefore, Klein teaches the additional limitation of claim 8.
Regarding claim 9, Liu discloses the plurality of expression vectors of claim 1 as discussed fully above and incorporated here. Liu further discloses each expression vector further contain a unique barcode sequence to identify individual constructs in a mixed population [Liu abstract]. However, Liu does not disclose each expression vector further comprises a cell barcode or a UMI sequence.
Klein discloses a massive parallel reporter assay in which reporter constructs are introduced into cells, transcribed RNA then captured and sequenced using primers containing both cell barcode and a unique molecular modifier (UMI) to enable normalization of transcriptional measurements across individual cells [Method section “RT–PCR, amplification and sequencing of RNA and DNA” and Supplementary Table 9]. Klein teaches that inclusion of cell-specific barcodes and UMI allows accurate quantification of reporter expression while correcting for amplification bias, thereby improving reproducibility of expression profiling across pooled constructs.
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify Liu’s barcode expression vectors to include additional barcodes such as cell-specific and/or UMI as taught by Klein. Klein teaches reporter transcripts in pooled assays benefit from additional barcode (e.g. UMI) during library preparation, since these additional identifiers allow correcting errors and accurately quantifying molecules by distinguishing original templates from PCR duplicates [Method section “RT–PCR, amplification and sequencing of RNA and DNA” and Supplementary Table 9]. As of the application’ s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to combine these teachings to extent pooled barcode expression assays from bulk to sing-cell quantitative readout and allow accurate cell-specific results, since both Liu and Klein employ barcoded library for quantifying construct expression. Hence, the proposed combination constitutes a predictable use of prior-art elements according to their established functions and would have been obvious to one of ordinary skill in the art at the time of filing. This reasoning is consistent with KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, A)
Regarding claim 11, Liu discloses the plurality of expression vectors of claim 1 as discussed fully above and incorporated here. Liu further discloses plasmids comprising unique sequence elements including promoters, ribosomal binding sites, elective markers, random DNA barcode [Fig. 1]. However, Liu does not disclose each expression vector further comprises a capture sequence or a polyadenylation signal.
Klein discloses each expression vector further comprises a capture sequence or a polyadenylation signal (e.g. high-throughput reporter assay in which enhancer variants are cloned into the STARR-seq reporter vector, a construct known in the art to contain a polyadenylation signal downstream of the reporter gene, Klein teaches sequencing reporter transcript using polyA-capture RNA-seq to qualify regulator activity [method section, “Design, barcoding and cloning of the enhancer library into the HSS vector”]. The STARR-seq backbone inherently provides a polyA tail for transcript stability and for capture during reverse transcription and sequencing).
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify Liu’s barcode expression vectors to include a capture sequence or polyadenylation signal as taught by Klein, in order to facilitate efficient transcript recovery and quantification if reporter expression to achieve to ensure accurate recovery of mRNA species for downstream readout. As of the application’ s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to combine these teachings to improve Liu’s assay performance by increasing analyte’s concentration and quality, since both Liu and Klein employ barcoded library for quantifying construct expression. Hence, the proposed combination constitutes a predictable use of prior-art elements according to their established functions and would have been obvious to one of ordinary skill in the art at the time of filing. This reasoning is consistent with KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, A)
Regarding claim 12, Liu and Klein disclose the plurality of expression vectors of claim 3 as discussed fully above and incorporated here. Klein further discloses the nucleic acid regulatory element and the cBC are linked. (e.g. enhancer [method section) is molecularly linked to a unique barcode [Fig. 1]. Klein’s system enables each regulatory element to be uniquely tracked by sequencing its associated barcode. Thereby correlating barcode counts with its regulatory). Therefore, Klein teaches the additional limitation of claim 12.
Regarding claim 13, Liu and Klein disclose the plurality of expression vectors of claim 12 as discussed fully above and incorporated here. Klein further discloses the nucleic acid regulatory element and cBC are linked through a process selected from synthesis, ligation, PCR, and any combination thereof. (e.g. cloning of thousands of enhancer variants into reporter vectors using the STARR-seq backbone, where in each enhancer is introduced into reporter construct through PCR amplification and ligation-dependance cloning [method section “Replicates, normalization and RNA/DNA activity scores”]). Therefore, Klein teaches the additional limitation of claim 13.
Liu et al., Klein et al., and Yamawaki et al.
Claim(s) 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (mSystems, 2018, Vol. 3, Issue 1) in view of Klein et al. (Nature Methods, 2020, Vol 17, pages 1083–1091) and Yamawaki et al. (BMC Genomics, 2021, 22:66)
Regarding claim 10, Liu and Klein disclose the plurality of expression vectors of claim 9 as discussed fully above and incorporated here. Klein further discloses the cell barcode comprises a UMI sequence (e.g. the reporter constructs can be analyzed at single cell resolution using RNA sequencing methods that utilize cell-specific barcodes and UMIs to accurately quantify transcript abundance for each cell [Klein Fig. 5, page 1089-1090]. However, Liu and Klein does not disclose 10x cell barcode and the UMI sequence comprises a 10x UMI sequence.
Yamawaki discloses 10x cell barcode and the UMI sequence comprises a 10x UMI sequence. (e.g. they tested variety of single-cell RNA-seq methods for cell profiling, including the Chromium 10x Genomics 3’ and 5’ workflows, which employ paired barcoding architecture of 10x barcode and 10x UMI sequence [page 15]. After comparing 10x Genomic with other single-cell RNA-seq methods, Yamawaki concludes that “10x Genomics methods had the highest cell recovery and mRNA detection sensitivity, making these techniques particularly suited to experiments with limited samples and experiments that require detection of genes with lower expression levels” [page 13, Discussion section]).
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the barcoded expression vector of Liu and Klein to include 10x Genomic single cell barcode technique, comprising 10x barcode and 10x UMI as disclosed by Yamawaki. Yamawaki further teaches 10x Genomics platforms provide superior cell recovery and RNA detection sensitivity across tested single-cell RNA-seq techniques. As of the application’ s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to adopt the 10x barcode and 10x UMI architectures, the proven most effective workflow, in context of Liu’s and Klein’s vector based reporter assay to achieve reliable single-cell identification and transcript quantification. This reasoning is consistent with KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143,A)
Regev et al., Melnikov et al., and Kivioja et al.
Claim(s) 14-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Regev et al. (US 20200362334 A1) in view of Melnikov et al.(Nature Biotechnology, 2012, Vol 30, No. 3, pages 271-277) and Kivioja et al.(Nature Methods, 2012, Vol 9, No. 1, pages 72-74).
Regarding claim 14, Regev discloses a method for determining individual activities of a plurality of nucleic acid regulatory elements, the method comprising: a. introducing the plurality of expression vectors into a population of cells (e.g. introduced a library of reporter constructs, each containing at least 1 construct per cell [paragraph 0023]); and b. performing single-cell RNA sequencing on the population of cells [paragraph 0023]. However, Regev does not explicitly disclose each expression vector of the plurality comprises a first identifying nucleic acid barcode (rBC) uniquely associated with the individual expression vector; and c. quantifying expression of cBC and/or rBC in an individual cell; wherein the amount of each cBC detected indicates the activity of the associated regulatory element in the cell and the amount of each rBC detected indicates the number of expression vectors comprising the associated regulatory element in the cell.
Melnikov discloses each expression vector of the plurality comprises a first identifying nucleic acid barcode (rBC) uniquely associated with the individual expression vector (e.g. a massively parallel assay in which each expression construct includes a sequence tag that is used to quantify both construct abundance and regulatory activity driven by the upstream enhancer variants. In single-hit MPRA, Melnikov teaches RNA count of each construct-specific barcode strategy enable both quantify and correct experimental noise [Figure 1, page 272 “Experimental design and mutagenesis strategies”]); and c. quantifying expression of cBC and/or rBC in an individual cell; wherein the amount of each cBC detected indicates the activity of the associated regulatory element in the cell (e.g. in multi-hit sampling, distinct enhancer variants each linked to a single barcode, enable individual enhancer variants to be assayed in in pooled expression assay. However, multi-hit sampling results in less accurate measurements for individual variants. [Figure 1, page 272 “Experimental design and mutagenesis strategies”]). and the amount of each rBC detected indicates the number of expression vectors comprising the associated regulatory element in the cell (e.g. In single-hit MPRA, Melnikov teaches RNA count of each construct-specific barcode strategy enable both quantify and correct experimental noise [Figure 1, page 272 “Experimental design and mutagenesis strategies”]. To estimate the activity of each enhancer variant, researchers compared the median of its mRNA/plasmid tag ratios to those of the corresponding Wild Type enhancer.[“Analysis of single-hit scanning variants “ in the “Online Method” section].
Kivioja further reinforces the important of normalizing sequencing data by adding unique molecular identifiers (UMIs) to genome-scale mRNA sequencing. These identifiers allow downstream sequencing readouts to be traced back to the correct construct [abstract]. Kivioja teaches amplification and sequencing introduce biases and potential errors that can lead to incorrect data interpretation if not addressed, and the use of UMI enable correction of experimental noise, normalizing read counts and accurate determination of each construct’s abundance in mixed population assays [page 72].
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify Regev’s single cell barcoded reporter system to incorporate the barcode-based normalization and data-correction strategies taught by Melnikov and Kivioja. Regev already teaches introducing the plurality of expression barcoded-vectors into a cells and performing single-cell sequencing. However, Regev does not explicitly disclose the claimed method of distinguishing construct identity and/or correcting sequencing and amplification errors. Melnikov provides solution for associating each regulatory element with a barcode, where RNA quantification informs the activity of linked enhancer. Melnikov also teaches a separate identification barcode for tracking individual constructs across pooled assays and showed barcode-based quantification allow accurate data readout despite experimental noise. Kivioja further discloses that adding a unique identification barcode to each template enable normalization of read counts, correction of amplification bias. As of the application’ s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to incorporating Melnikov’s barcoded-vectors and Kivioja’s normalization barcoding technique into the Regev’s high resolution single-cell sequencing to (i) quantitatively determine regulatory activity in individual cell and (ii) accurately quantify each construct’s transcripts in pooled assays. The combination displays applying established barcoding and normalization technique, which is already used in large pooled reporter assays, to Regev’s single cell sequencing to obtain more reliable data. This combination is consistent with KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, A).
Regarding claim 15, Regev, Melnikov, and Kivioja disclose the method of claim 14 as discussed fully above and incorporated here. Regev further discloses method comprising generating a scRNAseq profile for the individual cell, wherein the scRNAseq profile identifies the cell type of the individual cell (e.g. sequencing includes whole transcriptome analysis [paragraph 0034 and 0035]). Therefore, Regev teaches the additional limitation of claim 15.
Regarding claim 16, Regev, Melnikov, and Kivioja disclose the method of claim 14 as discussed fully above and incorporated here. Regev further discloses the population of cells comprises cells in different biological states, the different biological states comprise different stages of cell cycle, different subpopulations of same cell type, or a combination thereof (e.g. analyze heterogeneity of cell populations including cells of different states [paragraph 0869]). Therefore, Regev teaches the additional limitation of claim 16.
Regarding claim 17, Regev, Melnikov, and Kivioja disclose the method of claim 14 as discussed fully above and incorporated here. Regev further discloses the population of cells comprises multiple cell types (e.g. analyze heterogeneity of cell populations including cells of different states [paragraph 0869]). Therefore, Regev teaches the additional limitation of claim 17.
Regarding claim 18, Regev, Melnikov, and Kivioja disclose the method of claim 14 as discussed fully above and incorporated here. Melnikov further disclose normalizing the activity of the regulatory element to the number of expression vectors comprising the regulatory element in the cell. (e.g. To estimate the activity of each enhancer variant, researchers compared the median of its mRNA/plasmid tag ratios to those of the corresponding Wild Type enhancer.[“Analysis of single-hit scanning variants “ in the “Online Method” section]). Therefore, Melnikov teaches the additional limitation of claim 18.
Regarding claim 19, Regev discloses a method for identifying a regulatory element having cell type-specific activity, the method comprising: a. introducing the plurality of expression vectors into a population of cells (e.g. introduced a library of reporter constructs, each containing at least 1 construct per cell [paragraph 0023]), b. performing single-cell RNA sequencing on the population of cells [paragraph 0023]; d. generating a scRNAseq profile for the individual cell, wherein the scRNAseq profile identifies the cell type of the individual cell (e.g. sequencing includes whole transcriptome analysis [paragraph 0034 and 0035]); and e. determining the regulatory element to have cell type-specific activity if the activity of the regulatory element differs substantially between at least two cell types. (e.g. analyze heterogeneity of cell populations including cells of different states [paragraph 0869]). However, Regev does not explicitly disclose a. each expression vector of the plurality comprises a first identifying nucleic acid barcode (rBC) uniquely associated with the individual expression vector; and c. quantifying expression of cBC and/or rBC in an individual cell; wherein the amount of each cBC detected indicates the activity of the associated regulatory element in the cell, and the amount of each rBC detected indicates the number of expression vectors comprising the associated regulatory element in the cell.
Melnikov discloses each expression vector of the plurality comprises a first identifying nucleic acid barcode (rBC) uniquely associated with the individual expression vector (e.g. a massively parallel assay in which each expression construct includes a sequence tag that is used to quantify both construct abundance and regulatory activity driven by the upstream enhancer variants. In single-hit MPRA, Melnikov teaches RNA count of each construct-specific barcode strategy enable both quantify and correct experimental noise [Figure 1, page 272 “Experimental design and mutagenesis strategies”]); and c. quantifying expression of cBC and/or rBC in an individual cell; wherein the amount of each cBC detected indicates the activity of the associated regulatory element in the cell (e.g. in multi-hit sampling, distinct enhancer variants each linked to a single barcode, enable individual enhancer variants to be assayed in in pooled expression assay. However, multi-hit sampling results in less accurate measurements for individual variants. [Figure 1, page 272 “Experimental design and mutagenesis strategies”]). and the amount of each rBC detected indicates the number of expression vectors comprising the associated regulatory element in the cell (e.g. In single-hit MPRA, Melnikov teaches RNA count of each construct-specific barcode strategy enable both quantify and correct experimental noise [Figure 1, page 272 “Experimental design and mutagenesis strategies”]. To estimate the activity of each enhancer variant, researchers compared the median of its mRNA/plasmid tag ratios to those of the corresponding Wild Type enhancer.[“Analysis of single-hit scanning variants “ in the “Online Method” section].
Kivioja further reinforces the important of normalizing sequencing data by adding unique molecular identifiers (UMIs) to genome-scale mRNA sequencing. These identifiers allow downstream sequencing readouts to be traced back to the correct construct [abstract]. Kivioja teaches amplification and sequencing introduce biases and potential errors that can lead to incorrect data interpretation if not addressed, and the use of UMI enable correction of experimental noise, normalizing read counts and accurate determination of each construct’s abundance in mixed population assays [page 72].
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify Regev’s single cell barcoded reporter system to incorporate the barcode-based normalization and data-correction strategies taught by Melnikov and Kivioja. Regev already teaches introducing the plurality of expression barcoded-vectors into a cells, performing single-cell sequencing, generating a scRNAseq profile. However, Regev does not explicitly disclose the claimed method of distinguishing construct identity and/or correcting sequencing and amplification errors. Melnikov provides solution for associating each regulatory element with a barcode, where RNA quantification informs the activity of linked enhancer. Melnikov also teaches a separate identification barcode for tracking individual constructs across pooled assays and showed barcode-based quantification allow accurate data readout despite experimental noise. Kivioja further discloses that adding a unique identification barcode to each template enable normalization of read counts, correction of amplification bias. As of the application’ s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to incorporating Melnikov’s barcoded-vectors and Kivioja’s normalization barcoding technique into the Regev’s high resolution single-cell sequencing to (i) quantitatively determine regulatory activity in individual cell and (ii) accurately quantify each construct’s transcripts in pooled assays. The combination displays applying established barcoding and normalization technique, which is already used in large pooled reporter assays, to Regev’s single cell sequencing to obtain more reliable data. This combination is consistent with KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, A).
Regarding claim 20, Regev discloses a method for identifying a regulatory element having cell type-specific activity, the method comprising: a. introducing the plurality of expression vectors into a population of cells (e.g. introduced a library of reporter constructs, each containing at least 1 construct per cell [paragraph 0023]); b. performing single-cell RNA sequencing on the population of cells [paragraph 0023]; c. calculating the variance in activity of the regulatory element across the population of cells (e.g. analyze heterogeneity of cell populations including cells of different states [paragraph 0869]). However, Regev does not explicitly disclose a. each expression vector of the plurality comprises a first identifying nucleic acid barcode (rBC) uniquely associated with the individual expression vector; and c. quantifying expression of cBC and/or rBC in an individual cell; wherein the amount of each cBC detected indicates the activity of the associated regulatory element in the cell, and the amount of each rBC detected indicates the number of expression vectors comprising the associated regulatory element in the cell.
Melnikov discloses each expression vector of the plurality comprises a first identifying nucleic acid barcode (rBC) uniquely associated with the individual expression vector (e.g. a massively parallel assay in which each expression construct includes a sequence tag that is used to quantify both construct abundance and regulatory activity driven by the upstream enhancer variants. In single-hit MPRA, Melnikov teaches RNA count of each construct-specific barcode strategy enable both quantify and correct experimental noise [Figure 1, page 272 “Experimental design and mutagenesis strategies”]); and c. quantifying expression of cBC and/or rBC in an individual cell; wherein the amount of each cBC detected indicates the activity of the associated regulatory element in the cell (e.g. in multi-hit sampling, distinct enhancer variants each linked to a single barcode, enable individual enhancer variants to be assayed in in pooled expression assay. However, multi-hit sampling results in less accurate measurements for individual variants. [Figure 1, page 272 “Experimental design and mutagenesis strategies”]). and the amount of each rBC detected indicates the number of expression vectors comprising the associated regulatory element in the cell (e.g. In single-hit MPRA, Melnikov teaches RNA count of each construct-specific barcode strategy enable both quantify and correct experimental noise [Figure 1, page 272 “Experimental design and mutagenesis strategies”]. To estimate the activity of each enhancer variant, researchers compared the median of its mRNA/plasmid tag ratios to those of the corresponding Wild Type enhancer.[“Analysis of single-hit scanning variants “ in the “Online Method” section].
Kivioja further reinforces the important of normalizing sequencing data by adding unique molecular identifiers (UMIs) to genome-scale mRNA sequencing. These identifiers allow downstream sequencing readouts to be traced back to the correct construct [abstract]. Kivioja teaches amplification and sequencing introduce biases and potential errors that can lead to incorrect data interpretation if not addressed, and the use of UMI enable correction of experimental noise, normalizing read counts and accurate determination of each construct’s abundance in mixed population assays [page 72].
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify Regev’s single cell barcoded reporter system to incorporate the barcode-based normalization and data-correction strategies taught by Melnikov and Kivioja. Regev already teaches introducing the plurality of expression barcoded-vectors into a cells and performing single-cell sequencing. However, Regev does not explicitly disclose the claimed method of distinguishing construct identity and/or correcting sequencing and amplification errors. Melnikov provides solution for associating each regulatory element with a barcode, where RNA quantification informs the activity of linked enhancer. Melnikov also teaches a separate identification barcode for tracking individual constructs across pooled assays and showed barcode-based quantification allow accurate data readout despite experimental noise. Kivioja further discloses that adding a unique identification barcode to each template enable normalization of read counts, correction of amplification bias. As of the application’ s effective filing date, one of ordinary skill in the art would have had a reasonable expectation of success and motivated to incorporating Melnikov’s barcoded-vectors and Kivioja’s normalization barcoding technique into the Regev’s high resolution single-cell sequencing to (i) quantitatively determine regulatory activity in individual cell and (ii) accurately quantify each construct’s transcripts in pooled assays. The combination displays applying established barcoding and normalization technique, which is already used in large pooled reporter assays, to Regev’s single cell sequencing to obtain more reliable data. This combination is consistent with sKSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, A).
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684