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 .
Status of The Claims
Claims 1-2, 4-8, and 11-22 are pending and under examination.
Claims 3, 9-10 are canceled.
The following Office Action is in response to Applicant's communication dated 05/26/2026.
Response to Amendments/Arguments
The claim listing on 05/26/2026 fails to comply with 37 CFR 1.121 because the claim listing does not contain the appropriate markings indicating matter added to the amended claims. For example, current claim 1 appear to have been substantially rewritten without clearly and consistently underline all the additions relative to the prior version of claim 1 (06/20/2023). In current claim 14, 19, and 20 certain language that was already added and underlined in prior version of claim 14, 19, and 20 respectively (06/20/2023) appear to remain underlined in the present claim listing, obscuring what changes are being made in the present amendment. Thus, the amendment could be considered non-responsive. In the interest of compact prosecution, the amendment at issue will not be considered non-responsive. However, any future responses failing to comply with 37 CFR 1.121 will be held non-responsive, and will not be considered.
Applicant amended independent claims 1 require additional elements a, b, and d, thus, overcame the previous anticipation rejection base on Liu.
Applicant amended independent claims 14 requires “a second identifying nucleic acid barcode (cBC) uniquely associated with each nucleic acid regulatory element of the plurality of nucleic acid regulatory elements, and wherein the complexity of rBC is sufficiently large such that the probability (P) of collision of at least one rBC-cBC combination in any individual cell within a population of cells is less than or equal to 0.01”, thus overcame the previous rejection base on Regev, Melnikov, and Kivioja.
Accordingly, rejection(s) and/or objection(s) not reiterated from previous office actions are hereby withdrawn. The following rejection(s) and/or objection(s) are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
The amendment to claim 14 requires Examiner to conduct a new search and apply new prior art. Although claims 19 and 20 do not depends on claim 14, they are directed to similar variations of the same subject matter. Therefore, a new rejections of claims 19 and 20 were prompted by the amendment to claim 14, finality is proper.
Applicant’s argument filed 05/26/2026 have been fully considered but are not persuasive for the reasons set forth below. While applicant has amended the claims and presented arguments traversing prior rejections, the amendments and arguments do not overcome the rejections as presently applied. Hence, the rejections maintained.
In response to applicant's argument that Klein does not teach incorporated two different types of barcodes, especially the rBC, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
New Claim Objections-Necessitated by Amendments
Claim 22 is objected to because of improper dependency from itself. For purposes of examination, the claim is interpreted to depend from claim 21.
New Claim Interpretation-Necessitated by Amendments
Claims 21 and 22 are interpreted as adding limited structural weight to the claimed plurality of expression vectors of claim 1. Claims 21 and 22 do not recite additional vector components, such as sequence arrangements, linkage, or additional physical features. Instead, Claims 21 and 22 recite how barcode complexity may be evaluated under particular cell transfection conditions. Hence, the collision probability limitation is given weight to the extent it requires sufficient barcode complexity in the claimed plurality vectors. The limitation does not distinguish over prior art vectors libraries that already include random/high complexity barcode pairs capable of producing same low collision probability under comparable conditions.
In another word, collision probability is understood as an analytical characterization of vector library rather than a structural limitation of vector composition.
Modified Claim Rejections - 35 USC § 103- Necessitated by Amendment
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.
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.
Claim(s) 1, 2, 4-8, and 11-13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (mSystems, 2018, Vol. 3, Issue 1, of record) in view of Klein et al. (Nature Methods, 2020, Vol 17, pages 1083 1091, of record).
Regarding claim 1 and 2, 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, wherein 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]). 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]).
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 claims 4 and 5, Klein further discloses each nucleic acid regulatory element is a genetic variant of a single nucleic acid regulatory element and differs from each other nucleic acid regulatory element of the plurality of different 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]).
Regarding claims 6 and 7, Klein further discloses the the regulatory element is a cis-regulatory element, which 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, a re 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] ).
Regarding claims 8, 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.l, method section "Library cloning"]. Klein teaches that each vector contains a regulatory sequence driving expression of reporter gene [Fig 11).
Regarding claims 11, Klein further 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 provides a polyA tail for transcript stability and for capture during reverse transcription and sequencing).
Regarding claims 12, 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).
Regarding claim 13, 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"]).
New Claim Rejections - 35 USC § 103- Necessitated by Amendment
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.
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.
Hrvatin et al. and Michlits et al.
Claim(s) 14, 19, and 20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hrvatin et al. (Elife 8." 2019, disclosed in IDS) in view of Michlits et al. (Nat Methods 14, 1191–1197 (2017))
Regarding claim 14, Hrvatin discloses 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, wherein each expression vector of the plurality comprises (e.g. pooled AAV library comprising gene regulatory elements (GRE) constructs, wherein pooled library is delivered into mouse’s visual cortex. Each GRE is paired with one or more unique barcodes, and the barcode(s) is/are inserted into the 3’ untranslated region of the GRE induced transcript. [page 3])
b. performing single-cell RNA sequencing (scRNAseq) on the population of cells;[abstract]
c. quantifying expression of cBC; wherein an amount of each cBC detected indicates the activity of the associated regulatory element in the cell (e.g. quantify the abundance of the GRE-paired barcodes in each nucleus’s transcriptome to determine GRE-driven levels of expression [pages 5-6])
However, Hrvatin does not disclose each expression vector of the plurality comprises a randomized nucleic acid barcode (rBC) uniquely associated with the individual expression vector; and c. quantifying rBC; wherein the amount of each rBC detected indicates the number of expression vectors comprising the associated regulatory element in the cell.
Michlits discloses the random barcode (unique molecular identifiers-UMI) associated with individual constructs in a pooled CRISPR screening library, wherein the combination between an sgRNA and a random barcode distinguishes independent members of the library sharing the same sgRNA identity [abstract]. Additionally, Michlits teaches that individual UMIs associated with same sgRNA are counted and grouped, hence provide number of UMIs per sgRNA [Data analysis in Method section].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to apply Michlits’s randomized UMI strategy to Hrvatin’s GRE barcoded reporter because Hrvatin already teaches using a GRE associated barcode to identify regulatory element and quantify GRE related expression by single cell RNA sequencing. Michlits teaches that adding a random barcode to a construct allows independent vectors with same primary identity to be counted and distinguished. The modification allows vector derived effect to be detected, reducing false positives from multiple infections, and improve reproducibility of pooled screening readout [Michlits’s discussion].
Further, since Michlits uses randomized UMIs to distinguish independent barcode-labeled events sharing same primary sequence (cBC), the complexity is result-effective variable. It would have been obvious of person of ordinary skill in the art to select rBC complexity sufficient to reduce collision of rBC-cBC combinations, including probability less than 0.01, because such low probability of duplicate would predictably improve accurate assignment of independent vectors-derived events sharing same cBC. The claimed threshold is an obvious matter of routine optimization of barcode complexity.
Regarding claim 15 and 17, Hrvatin discloses base on sequencing results from single-cell RNA-sequencing, nuclei were classified into ten cell types using graph-based clustering and expression of known marker genes [page 6].
Regarding claim 16, Hrvatin discloses the population of cells comprises cells in different biological states including different subpopulations of same cell type (e.g. the reference identifies GREs that drive expression in SST neuronal subsets/subpopulations [pages 5-6])
Regarding claim 18, Hrvatin discloses determining GRE activity from the abundance of GRE associated barcode transcripts. Michlits discloses assigned sgRNA, UMI sequences, and experimental indices to sequencing reads, grouping independent UMIs associated with same sgRNA, and normalize barcode counts in pooled screening analysis [Data analysis]. It would be obvious to normalize Hrvatin’s GRE activity measurement by number of randomized UMI for that same GRE because as Michlits mentioned, doing so would account for differences in vector copy number or delivery events, improve accuracy of measured regulatory element activity.
Regarding claim 19, claim 19 recites similar method steps of claim 14 in combination with claims 15 and 17, including introducing barcoded vectors, performing scRNA sequencing, quantifying cBC and/or rBC in individual cell, generating scRNA profile identify cell type and determine regulatory activity. Thus, claim 19 is rejected over Hrvatin in view of Michlits for same reasons discussed above with respect to claims 14, 15, and 17.
Regarding claim 20, claim 20 recites similar method steps of claim 14 including: introducing barcoded vectors, performing scRNA sequencing, quantifying cBC and/or rBC in individual cell. Thus, claim 20 is rejected over Hrvatin in view of Michlits for same reasons discussed above with respect to claims 14.
Liu et al. and Klein et al.
Claim(s) 21 and 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (mSystems, 2018, Vol. 3, Issue 1, of record) in view of Klein et al. (Nature Methods, 2020, Vol 17, pages 1083 1091, of record).
Regarding claim 21 and 22, Liu discloses incorporation a random 20-nucleotide DNA barcode into transposon delivery vectors and further discloses estimated barcode diversity library is more than more than 10 million unique DNA sequences [pages 2-6]. Since the probability of collision decreases as barcode complexity increases, rBC complexity is result effective variable. When Liu’s highly complex barcode used with Klein’s regulatory element barcodes, it would have been obvious of person of ordinary skill in the art to select sufficient barcode complexity to reduce collision of rBC-cBC combinations, including probability less than 0.01, because such low probability of duplicate would predictably improve accurate assignment of independent vectors-derived events sharing same cBC. The claimed threshold is an obvious matter of routine optimization of barcode complexity.
Conclusion
No claims are allowed
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, 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 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.
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.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
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