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 .
Office Action: Notice
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/16/2025 has been entered.
Claim Status
Claims 1, 13, 48 and 57 have been amended (12/16/2025). No new matter was added. Thus, claims 1, 6, 10-11, 13, 17, 19, 22-23, 29-30, 39-40, 46, 48, 57-58, 61-62 and 65 are under examination.
Priority
Claims are given a priority date of 2/11/2019, the effective filing date of US Provisional 62803829.
Rejections Withdrawn
Claim Rejections - 35 USC § 102
The rejection of claims 1, 6, 10-11, 13, 17, 19, 22-23, 29-30, 39-40, 46, 48, 57-58, 61-62 and 65 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Engreitz et al. (WO 2018064208 A1, published 4/5/2018) is withdrawn in view of Applicant’s amendments of claims 1 and 13 and as a result of further clarification set forth in Applicant’s Remarks (12/16/2025).
New Rejections
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 6, 10-11, 13, 17, 19, 22-23, 29-30, 39-40, 46, 48, 57-58, 61-62 and 65 are rejected under 35 U.S.C. 103 as being unpatentable over Engreitz et al. (WO 2018064208 A1, published 4/5/2018), in view of Risca et al. (“Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping”, Nature, published 2017).
Regarding claim 1, Engreitz teaches assembling a synthetic transcription activation and post-translational modification complex consisting of multiple distinct effector domains and modifying enzymes (i.e., a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a transposon/transposase), as modeled after natural transcription activation processes (Paragraph 320, lines 5-15; Paragraph 680, lines 1-5). Specifically, Engreitz teaches, for genotyping deletion clones, a first round of PCR using primers followed by a second nested PCR on this product to add sequencing tags and clone-specific barcodes for high-throughput sequencing (Paragraph 908, lines 1-5; Table 1). Engreitz further teaches that the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site); that is, a short
sequence recognized by the CRISPR complex where the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used (Paragraph 170, lines 1-5). Engreitz, for example, teaches that in the case of Agrobacterium-mediated transformation, the marker cassette may be adjacent to or between flanking T-DNA borders and contained within a binary vector (Paragraph 641, lines 1-5).
Engreitz teaches that the previously described method is used to detect a change in gene expression in the cell, where the genomic region of interest is selected as a region which is 5' or 3' of a gene of interest, the phenotypic change can be determined based on expression of the gene of interest (Paragraph 107, lines 3-8). Further, Engreitz teaches that the barcoded or tagged region is recognized by a modified nuclease (i.e., ligase, a repair protein, a methyltransferase, (viral) integrase, a recombinase, a transposase, an argonaute, a cytidine deaminase, a retron, a group II intron, a phosphatase, a phosphorylase, a sulfurylase, a kinase, a polymerase, an exonuclease) (Paragraphs 174-175), including codon optimization may be combined with NLS or NES fusions, catalytically inactive nuclease modifications or nickase mutants may be combined with fusions to functional (heterologous) domains (Paragraph 175, lines 1-5).
Further, Engreitz teaches that the methodology described above is further analyzed via 4C technology, where all the DNA fragments captured by the bait in the population of cells are simultaneously amplified via inverse PCR, using two bait-specific primers that amplify from circularized ligation products following the addition of different barcodes to each sample (Paragraph 336, lines 10-15; Paragraph 907, lines 1-5).
Regarding claim 6, Engreitz teaches that the previously described synthetic activation and modification complexes, including transposon/transposase modifying enzymes, involve performing a first round of PCR using primers spanning the region of interest (Figure 10B), on both sides of the region or terminals, followed by a second nested PCR on this product to add sequencing tags and clone-specific barcodes for high-throughput sequencing (Table 1; Paragraph 908, lines 1-8).
Regarding claim 10, Engreitz teaches a library comprising a plurality of unique CRISPR-Cas system guide sequences, including the synthetic modified nucleases or transposon/transposase enzymes, that are capable of targeting a plurality of target sequences in one or more given genomic regions (Paragraph 325, lines 1-5). Further, Engreitz teaches that these unique guide sequences include non-naturally occurring or engineered compositions, comprising one or more of the elements, including clone-specific barcodes for high-throughput sequencing required to ensure genomic perturbation (Paragraph 328, lines 1-5; Paragraph 908, lines 1-8). Engreitz further teaches that the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site); that is, a short
sequence recognized by the CRISPR complex where the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used and PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence) (Paragraph 170, lines 1-5).
Regarding claim 11, Engreitz teaches the synthetic modified nucleases or transposon/transposase as described previously, may be used in the methods, compositions, and kits according to the invention (Paragraph 323, lines 1-3).
Regarding claim 13, Engreitz teaches a method for isolating a specific feature or regulatory element in chromatin via chromosome conformation capture (3C) technology, which provides a tool to study the structural organization of a genomic region. 3C technology involves quantitative PCR-analysis of cross-linking frequencies between two given DNA restriction fragments, which gives a measure or map of their proximity in the nuclear space (Paragraph 335, lines 1-5). Further, Engreitz teaches that 3C technology involves in vivo formaldehyde cross-linking of cells and nuclear digestion of chromatin with a restriction enzyme for activation, followed by ligation of DNA fragments local to a specified feature that were cross-linked into one complex (Paragraph 335, lines 5-10). Engreitz also teaches that the ligation products are then quantified by PCR to provide a measure of interaction frequencies between selected DNA fragments (Paragraph 335, lines 10-15). Additionally, Engreitz teaches that the sequence is determined by PCR, hybridization of a probe and/or sequencing, for example by sequencing using high-throughput paired end sequencing (Paragraph 346, lines 25-35). Engreitz teaches that the previously described method is used for applications in location mapping, including a labeled nucleotide is captured with a specific binding agent that specifically binds a capture moiety, such as biotin, on the labeled nucleotide and the capture moiety is adsorbed on a surface for labeling, capturing and detecting locations of altered DNA (Paragraph 346, lines 1-10).
Regarding claims 17, 19, and 22-23, Engreitz teaches that the sample analyzed via the previously described mapping method include permeabilized nuclei, multiple nuclei, isolated nuclei, synchronized cells, (such at various points in the cell cycle, for example metaphase) or acellular (Paragraph 348, lines 20-25). Further, Engreitz teaches that the previously described sample is immobilized or permeabilized on a solid support, thereby isolating the target nucleic molecule of interest (Paragraph 346, lines 10-15). Engreitz also teaches that the cells are further lysed to release the cellular contents following cross-linking (Paragraph 349, lines 1-5).
Regarding claim 29, Engreitz teaches that the previously described chromatin mapping can be used to explore developmental lineages, T cells (Jurkat cell line), B cells (Bjab), and monocytes (U-937), via chromatin accessibility with ATAC-Seq and found that they are highly similar to ATAC-Seq profiles of primary immune cells (Figure 31; Paragraph 1027, lines 1-10).
Regarding claims 30 and 39, Engreitz teaches that the previously described enzyme includes any type of functional domain may suitably be used, such as without limitation
including functional domains having one or more of the following activities: (DNA or RNA)
methyltransferase activity, methylase activity, demethylase activity, DNA hydroxylmethylase
domain, histone acetylase domain, histone deacetylases domain, transcription or translation
activation activity, transcription or translation repression activity, transcription or translation
release factor activity, histone modification activity, nuclease activity, single-strand RNA
cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity,
double-strand DNA cleavage activity, nucleic acid binding activity, a protein
acetyltransferase, a protein deacetylase, a protein methyltransferase, a protein deaminase (Paragraph 180, lines 10-15).
Regarding claim 40, Engreitz teaches that the previously described chromatin mapping includes; producing chromatin state maps in many cell types, providing the data needed to identify and evaluate which epigenomic features can predict gene-RE connections to enable studies to (1) observe the effects on gene expression; (2) compare these data to genome-wide maps of chromatin state to identify features that specify and predict gene connections; and (3) apply these predictions across many cell types to interpret human genetic variation (Paragraph 1016, lines 1-10).
Regarding claim 46, Engreitz teaches that the previously described chromatin mapping method comprises tagging an expression product of the gene with an antibody, wherein the cells are selected/sorted based on a quantitative measure of antibody binding (Paragraph 113, lines 1-5).
Regarding claims 48 and 57, Engreitz teaches that the previously described systematic mapping of chromatin state and chromosome conformation across cell types has been used to identify putative regulatory elements (Figure 3; Paragraph 3, lines 1-5). Specifically, Engreitz teaches that involved enzyme or modified nuclease includes a fusion protein comprising
the nuclease itself and one or more additional domains, which may be fused C-terminally or
N-terminally to the nuclease, or alternatively inserted at suitable and appropriate sited
internally within the nuclease (preferably without perturbing its function, which may be an
otherwise, modified function, such as including reduced or absent catalytic activity, nickase
activity, etc.). any type of functional domain may suitably be used, such as without limitation
including functional domains having one or more of the following activities: (DNA or RNA)
methyltransferase activity, methylase activity, demethylase activity, DNA hydroxylmethylase
domain, histone acetylase domain, histone deacetylases domain, transcription or translation
activation activity, transcription or translation repression activity, transcription or translation
release factor activity, histone modification activity, nuclease activity, single-strand RNA
cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity,
double-strand DNA cleavage activity, nucleic acid binding activity, a protein
acetyltransferase, a protein deacetylase, a protein methyltransferase, and/or a protein deaminase (Paragraph 180, lines 1-15).
Regarding claim 58, Engreitz teaches that the previously described chromatin mapping method involves separating a targeted population or RNA sequence via adaptation to bind
to an adaptor protein comprising a transcription repression domain (i.e., KRAB domain, NuE domain, an NcoR domain, a SID domain, or a SID4X domain) (Paragraph 96, lines 1-5). Further, Engreitz teaches that two strategies can be pursued to obtain these DNA circles or specified populations via (Paragraph 337, lines 1-20). Engreitz teaches that this method therefore comprises the steps of: (a) providing a sample of cross-linked DNA; (b) digesting the cross-linked DNA with a primary restriction enzyme— such as a 4 bp or a 5 bp cutter; (c) ligating the cross-linked nucleotide sequences; (d) reversing the cross linking and (e) amplifying the one or more nucleotide sequences of interest using at least two oligonucleotide primers (each primer hybridizes to the DNA sequences that flank the nucleotide sequences of interest) followed by hybridization to an array in order to assist in determining the frequency of interaction between the DNA sequences (Paragraph 227, lines 10-20). Further, Engreitz teaches that the second strategy involves the previously described method, but instead of performing an amplification reaction with primers that are specific for the fragments that one wishes to analyze, an amplification is performed using oligonucleotide primer(s) which hybridize to a DNA sequence that flanks the nucleotide sequences of interest (Paragraph 338, lines 1-5).
Engreitz teaches that following each PCR round using primers spanning the deleted region (Figure10B), a second nested PCR on the established product or targeted region of interest included adding sequencing tags and clone-specific barcodes for high-throughput sequencing (Table 1; Paragraph 908, lines 1-20) via amplicons on an Illumina™ MiSeq (Illumina, San Diego, CA) (Paragraph 907, lines 1-5).
Regarding claim 61, Engreitz teaches a multiomic or multi-step analysis process for DNA sequence variation via combined modifications (Paragraph 175, lines 1-10) or an unbiased genome-wide search for DNA fragments that interact with a locus of choice (Paragraph 338, lines 1-5). Further, Engreitz teaches that codon optimization may be combined with specified fusions, catalytically inactive nuclease modifications or nickase mutants may be combined with fusions to functional (heterologous) domains and analyzed (Paragraph 175, lines 1-10). Specifically, Engreitz teaches that the amplified fragments of interest are labeled and optionally hybridized to an array, typically against a control sample containing genomic DNA digested with the same combination of restriction enzymes. 3C technology has therefore been modified such that all nucleotide sequences of interest that interact with a target nucleotide sequence are amplified with primers that are specific for the fragments that flank the nucleotide sequences of interest (Paragraph 338, lines 15-25).
Regarding claim 62, Engreitz teaches that the previously described genomic region of interest comprises advantageous methods involving a putative regulatory element of the genome
susceptible to one or more of histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation and/or DNA methylation (Paragraph 148, lines 1-15).
Regarding claim 65, Engreitz teaches that the sample undergoes shearing or cleavage
generating sticky ends (i.e., staggered cut with a 5' overhang, overhang of 1 to 5 nucleotides)
(Paragraph 169, lines 5-10).
However, Engreitz does not teach or suggest targeting specifically a transpose to a specific chromatin feature to non-destructively alter or label DNA in methodology related to chromatin mapping.
Risca teaches genome-wide chromatin mapping via ionized radiation-induced spatially correlated cleavage of DNA with sequencing (RICC-seq) to identify DNA-DNA contacts that are spatially proximal (Abstract) and composed of 20-70 bp in length (Chromatin fibre structure simulation: Paragraph 1). Further, Risca teaches the calculation of the enrichment of different RICC-seq fragment lengths via enrichment of specified clusters (i.e., open chromatic associated with assay for transposase-accessible chromatin with high-throughput sequencing) and transcriptionally repressed chromatin associated with experiment-related specifications (Figure 2A; Main: Paragraphs 4-8), including histone modifications associated with open chromatin-associated modifications or ATAC-seq peaks (Figure 2E and Extended Data Figure 5) that were later analyzed using Python code to obtain a track of transposase insertion density over the genome (Sequencing data processing and alignment: Paragraph 1).
It would have been obvious to a person of ordinary skill in the art at the time of the invention to combine the chromatin labeling and sequencing workflow of Engreitz with the spatial analysis techniques taught by Risca because both references are directed to the same technical problem of genome-wide chromatin mapping using sequencing data, and Risca provides a known method for extracting additional positional and structural information from sequencing results already generated by Engreitz’s methods. Incorporating Risca’s analytical approach into Engreitz’s workflow represents the application of a known sequencing-data interpretation technique to an existing chromatin mapping method in order to improve resolution and interpretability of chromatin feature localization.
Further, a person of ordinary skill in the art would have had a reasonable expectation of success in making this combination because both Engreitz and Risca rely on well-established molecular biology techniques, including enzymatic DNA modification, high-throughput sequencing, and computational analysis of sequencing reads. Risca further demonstrates that sequencing-delivered DNA fragment positions and distances correlate with chromatin structure, and Engreitz demonstrates that chromatin features can be enzymatically targeted and identified through sequencing. Accordingly, applying Risca’s spatial analysis framework to the sequencing output of Engreitz would have predictably yielded enhanced chromatin feature mapping without requiring undue experimentation or technical modification of the underlying sequencing workflow.
Applicant’s Response: The Applicant argues that the primary reference, Engreitz, does not disclose all elements of the claimed invention. Specifically, Engreitz fails to teach a synthetic transposon architecture or targeted enzymatic labeling workflow, but instead relies on CRISPR-mediated cleavage, restriction design, and inferential mapping. The Applicant further asserts that generic listings of enzymes in Engreitz do not teach or require localized, non-destructive DNA alteration within a defined nucleotide window or mapping by long-read sequencing. Accordingly, the Applicant argues that the rejection improperly relies on inherency and post-hoc reconstruction rather than an express disclosure in the sequential steps as arranged.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered and are found to be partially persuasive, as discussed below.
The new USC 103 rejection, as written above, addresses the Applicant’s remarks, as well as amended independent claims 1 and 13. The combination of Engreitz with Risca to account for the sequencing-based spatial analysis and chromatin mapping functionality. The rejection further articulates a clear reason to combine, through improving positional resolution of chromatin feature mapping, and a reasonable expectation of success.
Specifically, the amendments and Applicant’s Remarks (12/16/2025) clarify that the instant claims require targeted, non-destructive enzymatic alteration or labeling of DNA at a defined positional relationship to a chromatin feature, rather than cleavage-based disruption for inferential mapping as taught by Engreitz. The Applicant further clarified that the claimed methods rely on direct positional mapping using sequencing of labeled DNA, and therefore distinguishing over Engreitz’s CRISPR-based screening. These clarifications resolved the anticipation deficiencies by making direct structural and operational limitations that are not disclosed in Engreitz, alone.
Rather, the new 103 rejection addresses these clarified distinctions by expressly identifying where Engreitz provides enzymatic targeting and sequencing-based chromatin analysis, and where Risca supplies the sequencing-based spatial interpretation needed to account for positional mapping, together with a reason to combine and a reasonable expectation of success.
To overcome the 103 rejections, the Examiner recommends further amending the claims to directly exclude cleavage or fragmentation-based spatial inference or alternatively submitting evidence showing that a skilled artisan would not have reasonably expected Risca’s spatial analysis techniques to be compatible with Engreitz’s enzymatic targeting approaches.
Conclusions
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/
Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684