DETAILED ACTION
The amendment filed on 09/25/2025 has been entered. No new matter has been added.
Claims 1, 3-6, 10, and 11 are amended in the claim set filed on 09/25/2025.
Claims 1-12 in the claim set filed on 09/25/2025 are pending and currently under examination.
Response to the Arguments
Objections to the Drawings in the previously mailed non-final have been withdrawn in light of applicants Drawings amendments.
Objections to the Specification in the previously mailed non-final are maintained in the absence of a IDS being filed.
Applicant’s arguments regarding previous rejection(s) of claim(s) 5-7 and 11 under 35 U.S.C. 112 have been fully considered and are persuasive. The 35 U.S.C. 112 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 5-6.
Applicant’s arguments regarding previous rejection(s) of claim(s) 1-8 and 12 under 35 U.S.C. 102 have been fully considered and are not persuasive. Applicant’s argument on Pg. 6-7, states that “Loveless because this reference does not teach or disclose all of the elements of claim 1. For example, Loveless does not disclose a "DNA-synthesis based recording system ... wherein concentration of nucleotides in the single cell is recorded," as recited in claim 1. Loveless relates to recording responses based on the dose of exposure to hypoxia as a stimulus. See Loveless, Introduction. Nothing in Loveless addresses a recording system that records concentration of nucleotides in a single cell.” The 35 U.S.C. 102 rejections documented in the previously mailed non-final have been maintained and revised (documented below on Pg. 4-9) in light of applicants claim amendments and arguments on Pg. 6-7.
Applicant’s arguments regarding previous rejection(s) of claim(s) 9-11 under 35 U.S.C. 103 have been fully considered and are not persuasive. Applicant’s argument on Pg. 7-8, states that “As noted in the previous section, Loveless does not teach or suggest a DNA-synthesis based recording system "wherein concentration of nucleotides in the single cell is recorded." Kalhor does not remedy the deficiencies of Loveless. … However, Kalhor does not teach or suggest examination of nucleotide concentrations in the cells, much less a "DNA-synthesis based recording system ... wherein concentration of nucleotides in the single cell is recorded." Nor does Kalhor provide a reasonable basis for reasonably expecting such a recording system. Further, the instant application provides unexpected results of a DNA-synthesis based recording system that records concentration of nucleotides. … thus providing a single-cell level resolution." Id. This provides a DNA-synthesis based recording system in a single-cell that is not addressed by the cited arts, alone or in combination.” The 35 U.S.C. 103 rejections documented in the previously mailed non-final have been maintained (documented below on Pg. 9-11) in light of applicants claim amendments and arguments on Pg. 7-8.
As necessitated by amendment to the claim, new grounds of rejection for claim 12 under Non-Statutory Double Patenting is documented below, in this office action on Pg. 12-15.
The rejections for claims 1-12 are documented below in this Final Office Action are necessitated by claim amendments filed on 09/25/2025.
Priority
This application claims priority to U.S. Provisional Application No. 63/157,521, filed March 5, 2021. Accordingly, the priority date of instant claims is determined to be March 5, 2021, the filing date of U.S. Provisional Application No. 63/157,521.
Specification
The listing of references in the specification is not a proper information disclosure
statement. The specification filed on 03/04/2022 includes a list of references on pages 12-13. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted
for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be
incorporated into the specification but must be submitted in a separate paper." Therefore,
unless the references have been cited by the examiner on form PTO-892, they have not been
considered.
Claim Rejections - 35 USC § 102
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.
Claims 1-8 and 12 remain/are rejected under 35 U.S.C. 102 (a)(1) as being anticipated by Loveless et al. (“Loveless”; (2019). DNA writing at a single genomic site enables lineage tracing and analog recording in mammalian cells. bioRxiv, 639120. Published Sep. 29, 2019).
Loveless disclosed a new type of DNA recorder that acts primarily by writing new DNA. The system of Loveless, called CHYRON (Cell HistorY Recording by Ordered iNsertion), inserts random nucleotides at a single locus in temporal order in vivo and can be applied as an evolving lineage tracer as well as a recorder of user-selected cellular stimuli. As a stimulus recorder, Loveless showed that when the CHYRON machinery was placed under the control of a stress-responsive promoter, the frequency and length of writing reflected the dose and duration of the stress. With further engineering of CHYRON’s components to increase writing efficiency, CHYRON should lead to single-cell-resolution recording of lineage and other information through long periods of time in complex animals or tumors, advancing the pursuit of a full picture of mammalian development (Summary).
Regarding claim 1, Loveless teaches a system comprising “a new type of DNA recorder that acts primarily by writing new DNA” and “amount written” (Summary). Loveless teaches a system comprising “uniqueness of CHYRON as a DNA recorder based on writing DNA” (Pg. 8, Current and future capabilities of CHYRON, Para. 1; Figure 1A see below). Loveless teaches a system comprising a Cas9 nuclease with an hgRNA and a DNA polymerase, terminal deoxynucleotidyl transferase (TdT) (Pg. 3, Para. 2; Figure 1, Design of the CHYRON locus; Figure 1A see below). Loveless teaches a system comprising “the number of reads for each unique insertion sequence in the well was determined and divided by the total to give the percentage abundance of that insertion” (Figure S4 legend). Loveless teaches a system comprising a “single-cell-resolution lineage reconstruction” (Pg. 11, Further analysis of successful reconstruction, Para. 1; Fig 1A see below). Thus, Loveless teaches a system comprising a DNA-synthesis based recording system, comprising a Cas, a homing guide RNA (hgRNA), and a terminal deoxynucleotidyl transferase (TdT), wherein the Cas, the hgRNA, and the TdT are all comprised within a single cell, and wherein concentration of nucleotides in the single cell is recorded.
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The teachings of Loveless are documented above in the rejection of claim 1 under 35 U.S.C. 102 (a)(1).
Regarding claim 2, Loveless teaches a system comprising a Cas9 (e.g., Pg. 3, Para. 2). Thus, Loveless teaches a system wherein the Cas is Cas9.
Regarding claim 3, Loveless teaches a system wherein the hgRNA repeatedly directs Cas9 to cut its own locus at a defined location relative to the PAM (e.g., Figure 1A (Shown below); Pg. 3, Para. 2, Figure 1, Design of the CHYRON locus). Thus, Loveless teaches a system wherein the Cas forms a complex with hgRNA and targets a DNA locus of the hgRNA.
Regarding claim 4, Loveless teaches a system wherein TdT-mediated insertional
mutagenesis generated an average insertion length of 2.9 bp (5.2 bits) per round (e.g., Pg. 3, Results and Discussion, Para. 2; Fig. 1A see above). Thus, Loveless teaches a system wherein the DNA locus of the hgRNA is diversified after each edit.
Regarding claim 5, Loveless teaches a system wherein a DNA polymerase, terminal deoxynucleotidyl transferase (TdT), capable of efficiently writing random nucleotides (nts) at Cas9-induced DSBs. Loveless teaches a system comprising a recorder by constructing a mutating DNA barcode called CHYRON (Cell HistorY Recording by Ordered iNsertion) (e.g., Pg. 3, Para. 2; Figure 1A, shown above). Loveless teaches a method to barcode individual cells containing the integrated CHYRON locus, unique molecular identifiers (UMIs) of 20 degenerate nucleotides were incorporated (e.g., Pg. 10, Para. 4). Thus, Loveless teaches a system wherein the TdT is directed to double-stranded breaks created by Cas at the hgRNA sites, and TdT adds at least one nucleotide at the double-stranded breaks, and wherein the at least one nucleotide optionally comprises a barcode.
Regarding claims 6 and 7, Loveless teaches a system wherein “CHYRON combines a Cas9 nuclease with an hgRNA and a DNA polymerase, terminal deoxynucleotidyl transferase (TdT), capable of efficiently writing random nucleotides (nts) at Cas9-induced DSBs. These newly-written nts are then incorporated into the repaired DSB to produce a durable insertion mutation consisting of random base pairs (bps)” (Pg. 3, para. 2; See Fig. 1 above). “efficiently writing random nucleotides (nts) at Cas9-induced DSBs” reads on “depends on the concentration of nucleotides in the single cell” and “base additions can be altered by altering the nucleotide concentration”. Thus, Loveless teaches a system wherein identity of the nucleotide added by the TdT depends on the concentration of nucleotides in the single cell and the TdT-directed base additions can be altered by altering the nucleotide concentration.
Regarding claim 8, Loveless teaches a system wherein CHYRON combines Cas9 nuclease with an hgRNA and a DNA polymerase, terminal deoxynucleotidyl transferase (TdT), capable of efficiently writing random nucleotides (nts) at Cas9-induced DSBs (e.g., Pg. 3, Para. 2). Loveless teaches a system wherein CHYRON is able to diversify a very compact recording locus, consisting of a single site that is repeatedly modified, so that the locus can bear a unique sequence in each of tens of thousands of cells. Loveless teaches a system wherein CHYRON records information by generating an ordered accumulation of random insertions. (e.g., Pg. 7, section “Current and future capabilities of CHYRON”, Para. 1). The information recorded (i.e., accumulation of insertions) is interpreted as an output signal. Thus, Loveless teaches a system wherein a change in TdT-based nucleotide incorporation into a hgRNA double-stranded break is defined as an output signal.
Regarding claim 12, Loveless teaches a method wherein the activity of such systems is linked to the presence of an arbitrary biological stimulus, accumulated mutations become a record of the strength and duration of exposure to the stimulus and when activity is constitutive, accumulated mutations capture lineage relationships among individual cells (Pg. 2 Introduction, Para. 2). Loveless teaches a method to test stimulus recording with CHYRON, wherein linked insertional mutagenesis at a CHYRON locus to hypoxia, which triggers adaptive responses that affect a wide range of cellular behaviors, including several, such as migration and invasion, that are important for tumor evolution and metastasis. Loveless teaches a method wherein they transfected 293T-CHYRON20 and 293T-CHYRON16 (which bears a 16-nt-spacer hgRNA integrated at the AAVS1 locus, as in CHYRON16i but without insulators) with this construct, and then exposed them to three different concentrations of the hypoxia mimic DMOG for five different durations. Loveless teaches in both cell lines, the proportion of the population bearing insertions increased with dose and duration of DMOG treatment (Figures 6B). Loveless teaches CHYRON is capable of recording exposure to stimuli in a manner that is digital (Figures 6B) or analog (Figure 6C), where the latter of these modes can in principle provide information on the experience of each single cell (e.g., Pg. 7, Para. 2). “293T” cells are considered an organic environment. Hypoxia is considered a variable. Thus, Loveless teaches a method establishing connections between cells, comprising exposing at least two cells that each comprise a DNA-synthesis based recording system according to claim 1 to an organic environment comprising deoxyribonucleotide triphosphates (dNTPs) and a variable, allowing the TdT to add dNTPs to a DNA substrate, and isolating the DNA substrate; wherein the dNTP content of the DNA substrate corresponds to the concentration of the variable in the organic environment.
Response to Arguments
Applicant' s arguments filed 09/25/2025 (Pg.6-7) with respect to claim 1-8 and 12 have been considered but are not persuasive. To clarify some instances argued in the response filed 09/25/2025 see responses to each argument made by Applicant below:
Applicants’ argument: “Loveless does not disclose a "DNA-synthesis based recording system ... wherein concentration of nucleotides in the single cell is recorded," as recited in claim 1.” (Pg. 6)
Response: Applicant's argument filed 09/25/2025 has been fully considered but is not persuasive because, as stated in the revised rejection under 35 U.S.C. 102 (documented above on Pg. 5-6) towards claim 1, Loveless does teach DNA-synthesis based recording system ... wherein concentration of nucleotides in the single cell is recorded. In short, Loveless teaches a system comprising “a new type of DNA recorder that acts primarily by writing new DNA” and “amount written” (Summary; Fig. 1A).
Applicants’ argument: “Loveless does not teach or disclose all of the elements of claim 1, much less the combination of elements of claims 2-8 and 12.” (Pg. 7)
Response: Applicant's argument filed 09/25/2025 has been fully considered but is not persuasive. Loveless does teach the elements of claim 1. Please see the response to the argument documented above in the rejection of claim 1 under 35 U.S.C. 102 (a)(1).
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.
Claims 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over Loveless et al. (“Loveless”; (2019). DNA writing at a single genomic site enables lineage tracing and analog recording in mammalian cells. bioRxiv, 639120. Published Sep. 29, 2019) in view of Kalhor et al. (“Kalhor” (2017). Rapidly evolving homing CRISPR barcodes. Nature methods, 14(2), 195–200).
The teachings of Loveless are documented above in the rejection of claims 1-8 and 12 under 35 U.S.C. 102 (a)(1).
Loveless does not teach the output signal is detectable with in situ sequencing, the cell is a neuron and/or the neuron is within the brain of a living mammal.
Kalhor discloses a system for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9–hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR–Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping. (Abstract).
Regarding claim 9, Kalhor teaches a system wherein in situ sequencing is a highly desirable method for barcode retrieval as it allows lineage information to be extracted without loss of histological information such as position and cell type (e.g., Pg. 197, Col. 2 Para. 3.; Fig. 1). Thus, Kalhor teaches a system wherein the output signal is detectable with in situ sequencing.
Regarding claim 10-11, Kalhor suggests a system wherein adequate barcoding of mouse neurons, which is essential for some of the proposed brain-mapping projects (e.g., Pg. 198, Col. 2 Para. 1). Thus, Kalhor suggests a system wherein the cell is a neuron, and the neuron is within the brain of a living mammal.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to include the in situ sequencing of output signal, the cell is a neuron, and is within the brain of a living mammal as taught/suggested by Kalhor to CHYRON DNA recorder system as taught by Loveless to have yielded the predictable result of sequencing within a cell, neuron, or brain of a live mammal. It would be obvious to the ordinary artisan to include the in situ sequencing of output signal, the cell is a neuron and is within the brain of a living mammal to Kalhor to the CHYRON DNA recorder system of Loveless with the reasonable expectation of improving the recording of cellular lineage history and mapping of cells and tissues such as the brain.
Response to Arguments
Applicant's arguments filed 09/25/2025 (Pg. 8) with respect to claims 9-11 have been considered but are not persuasive. Please see response to arguments documented above in the rejection of claims 1-8 and 12 under 35 U.S.C. 102 (a)(1). To clarify some instances argued in the response filed 09/25/2025 see responses to each argument made by Applicant below:
Applicants’ argument: “As noted in the previous section, Loveless does not teach or suggest a DNA-synthesis based recording system "wherein concentration of nucleotides in the single cell is recorded." Kalhor does not remedy the deficiencies of Loveless.” (Pg. 7)
Response: Applicant's argument filed 09/25/2025 has been fully considered but is not persuasive. Loveless does teach the elements of claim 1. Please see the response to the argument documented above in the rejection of claim 1 under 35 U.S.C. 102 (a)(1).
Applicants’ argument: “Kalhor does not teach or suggest examination of nucleotide concentrations in the cells, much less a "DNA-synthesis based recording system ... wherein concentration of nucleotides in the single cell is recorded." Nor does Kalhor provide a reasonable basis for reasonably expecting such a recording system.” (Pg. 7)
Response: Applicant's argument filed 09/25/2025 has been fully considered but is not persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicants’ argument: “the instant application provides unexpected results of a DNA-synthesis based recording system that records concentration of nucleotides… thus providing a single-cell level resolution. Id. This provides a DNA synthesis based recording system in a single-cell that is not addressed by the cited arts, alone or in combination." (Pg. 7-8)
Response: Applicant's argument filed 09/25/2025 has been fully considered but is not persuasive. In response to applicant's argument, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). Furthermore, Loveless does provide a system that would be expected to have single cell level resolution recording. (Pg. 2 Summary; Pg. 6 last para.; Pg. 7 para. 1 and 2; Pg. 8 para. 1)
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 12 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of copending U.S. Patent App. No. 17/420,606. (filed on 07/02/2021) in view of Loveless et al. (“Loveless”; (2019). DNA writing at a single genomic site enables lineage tracing and analog recording in mammalian cells. bioRxiv, 639120. Published Sep. 29, 2019). Although the claims at issue are not identical, they are not patentably distinct from each other because the instantly claimed invention is made obvious by the claims of copending Application No. 17/420,606 in view of Loveless.
Claim 1 of copending Application No. 17/420,606 is drawn to:
“1. A method of identifying a biological signal comprising exposing a template- independent DNA polymerase to an organic environment comprising deoxyribonucleotide triphosphates (dNTPs) and a variable, allowing the DNA polymerase to add dNTPs to a DNA substrate, and isolating the DNA substrate; wherein the dNTP content of the DNA substrate corresponds to the concentration of the variable in the organic environment.”
Instant claim 12 is directed to “A method of establishing connections between cells, comprising exposing at least two cells that each comprise a DNA-synthesis based recording system according to claim 1 to an organic environment comprising deoxyribonucleotide triphosphates (dNTPs) and a variable, allowing the TdT to add dNTPs to a DNA substrate, and isolating the DNA substrate; wherein the dNTP content of the DNA substrate corresponds to the concentration of the variable in the organic environment.”
The teachings of Loveless are documented above in the rejection of claims 1-8 and 12 under 35 U.S.C. 102 (a)(1).
Therefore, the invention as recited in claims 1 is prima facie obvious over the copending Application No. 17/420,606 in view of Loveless. One of ordinary skill in the art would have had a reasonable expectation of success given the lack of novelty. It would have been obvious to use a method of identifying a biological signal comprising exposing a template- independent DNA polymerase to an organic environment according to the limitations recited in claim 1 of the instant application based on claims 2 and 3 of copending Application No. 17/420,606 in view of Loveless et al.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
Applicant's arguments filed 09/25/2025 do not apply to the new grounds of rejections.
Conclusion of Response to Arguments
In view of the amendments, documented above in this Final Office Action are revised and new grounds of rejections as well as responses to arguments. No claims are in condition for allowance.
Conclusion
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 KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682