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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 02/27/2026 has been entered.
Claims 51 and 75 were amended in the claim set filed 02/27/2026.
Accordingly, claims 51-54 and 71-86 are pending and under consideration.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. The earliest effective filing date to which the instant application is entitled is 09/27/2019.
Status of Prior Objections/Rejections
RE: Claim Objections
►Claims 51 and 75 were previously objected to because for minor informalities.
The amendments to the instant claim set have obviated the basis of the objections of record. The objections of record are hereby withdrawn.
RE: Claim Rejections – 35 USC § 103
►Claims 51, 73-77, and 79-81 were previously rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (of record), in view of Ammar et al., 2012 (of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford), and Cadiñanos and Bradley, 2007 (of record), as evidenced by Chalmers and Kleckner, 1994 (of record).
►Claim 52 was previously rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (of record), in view of Ammar et al., 2012 (of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford), and Cadiñanos and Bradley, 2007 (of record), as evidenced by Chalmers and Kleckner, 1994 (of record) as applied to claim 51 above, and further in view of Nesmelova and Hackett, 2010 (of record) and Zhang et al., 2019 (of record).
►Claims 53, 54, and 77 were previously rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (of record), in view of Ammar et al., 2012 (of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford), and Cadiñanos and Bradley, 2007 (of record), as evidenced by Chalmers and Kleckner, 1994 (of record) as applied to claim 51 above, and further in view of Bhatt and Chalmers, 2019 (of record), as evidenced by Bouuaert and Chalmers, 2017 (of record).
►Claims 71 and 72 were previously rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (of record), in view of Ammar et al., 2012 (of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford), Cadiñanos and Bradley, 2007 (of record), and Bhatt and Chalmers, 2019 (of record), as evidenced by Chalmers and Kleckner, 1994 (of record) and Bouuaert and Chalmers, 2017 (of record) as applied to claim 54 above, and further in view of WO 2019/070762 (hereinafter Lee; of record), as evidenced by Sakamoto et al., 2017 (of record) and Jiang and Doudna, 2017 (of record).
►Claim 78 was previously rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (of record), in view of Ammar et al., 2012 (of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford), and Cadiñanos and Bradley, 2007 (of record), as evidenced by Chalmers and Kleckner, 1994 (of record) as applied to claim 51 above, and further in view of Li et al., 2016 (of record), as evidenced by Tsien, 1998 (of record).
►Claim 79 was previously rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (of record), in view of Ammar et al., 2012 (of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford), and Cadiñanos and Bradley, 2007 (of record), as evidenced by Chalmers and Kleckner, 1994 (of record) as applied to claim 51 above, and further in view of Adey et al., 2010 (of record), as evidenced by Pray, 2008 (of record).
►Claims 82-86 were previously rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (of record), in view of WO 2017/203267 A1 (hereinafter Oxford), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), Ammar et al., 2012 (of record), Cadiñanos and Bradley, 2007 (of record), Bhatt and Chalmers, 2019 (of record), and WO 2019/070762 (hereinafter Lee; of record), as evidenced by Chalmers and Kleckner, 1994 (of record) and Sakamoto et al., 2017 (of record).
Applicant has traversed the rejections of record, asserting that the methods of Oxford previously applied to the claimed transposase activation are in fact drawn to preparation of a helicase-not a transposase. In response, this is found persuasive.
However, further review of the art has necessitated new grounds of rejection, which are set forth below.
New/Maintained Grounds of Objection/Rejection
Specification
The disclosure is objected to because of the following informalities:
The instant specification references colors that are not present in the black and white drawings (see for example page 9, line 14). Should Applicant require color drawings, a petition must be filed under 37 CFR 1.84(a)(2), as set forth in the non-final rejection dated 05/05/2025.
Appropriate correction is required.
Claim Objections
Claim 82 is objected to because of the following informalities:
Claim 82 recites “a method of preparing a nucleic acid construct, comprising, in a reaction vessel:
inactivating a transposase…
contacting a target polynucleotide with…
(d) reactivating the inactivated transposase by…”,
which lists (a), (b), and (d), but skips sequential letter (c). In order to comport with standard linguistic conventions, it would be remedial to amend the instant claim language such that it recites (a), (b), and (c) rather than (a), (b), and (d).
Appropriate correction is required.
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.
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 51, 73-76, and 78-81 are rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (hereinafter Chavez; of record), in view of Cadiñanos and Bradley, 2007 (hereinafter Cadiñanos; of record), Strecker et al., 2019 (hereinafter Strecker; as cited in the IDS filed 07/07/2022), Klompe et al., 2019 (hereinafter Klompe; as cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford; of record), Carter et al., 2019 (hereinafter Carter), Ammar et al., 2012 (hereinafter Ammar; of record), and WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022).
With regard to claim 51, which recites “a method of preparing a nucleic acid construct for single molecule characterization, comprising, in a reaction vessel:
(a) inactivating a transposase by:…
(ii) providing a metal ion chelation agent…
(b) contacting a target polynucleotide with:
a polynucleotide-guided effector protein,
a guide polynucleotide,
the inactivated transposase,
and a transposable element comprising a modified polynucleotide;
wherein the polynucleotide-guided effector protein directs the inactivated transposase to a region of interest within the target polynucleotide;
(c) removing any transposase that is not bound to the polynucleotide-guided effector protein; and
(d) reactivating the inactivated transposase by:…
(iii) providing a solution comprising divalent ions;
wherein the reactivated transposase inserts the transposable element into the polynucleotide; thereby producing a nucleic acid construct for single molecule characterization,” as previously set forth, Chavez summarizes advances in site-programmable transposition (reported in Klompe and Strecker), specifically disclosing that Strecker engineered a system to deliver a helper plasmid containing TnsB, TnsC, TniQ, Cas12k, a tracrRNA, and a synthetic crRNA; a donor plasmid containing DNA flanked by transposition machinery sites; and a target plasmid library (page 207, column 1, paragraph 1). This system thus delivers the instantly claimed polynucleotide-guided effector protein (Cas12k), a guide polynucleotide (tracrRNA and crRNA), transposase (TnsB), and transposable element (donor plasmid containing DNA flanked by transposition machinery sites) to a target polynucleotide (the target plasmid library). As previously set forth, Chavez discloses that the Cas12k taught therein must associate with TniQ, TnsB, and TnsC in order for this system to function properly (page 207, column 3, paragraph 1; Figure 1) .However, Chavez does not disclose the inactivation of said transposase, the modified polynucleotide within said transposable element, the removal of any unbound transposase, the reactivation of said transposase, the insertion of a modified polynucleotide, or its applicability to single molecule characterization, as instantly claimed. However, these deficiencies are cured by various secondary references, as set forth below.
Regarding the inactivation and reactivation of the claimed transposase, as previously set forth, Cadiñanos discloses an inducible piggyBac transposon system (abstract), in which piggyBac activity is regulated by the presence or absence of 4- hydroxytamoxifen (page 4, column 2, paragraph 2-page 5, column 1, paragraph 1; Figure 4). Cadiñanos explicitly discloses that this inducible system was developed to prevent excessive and uncontrolled transposition mediated by active transposases that results in genomic instability such as inversions, deletions, and translocations by facilitating temporal regulation of transposition activity (page 1, column 2, paragraph 3; page 6, column 2, paragraph 1-page 7, column 1, paragraph 1). Thus, while the disclosure of Cadiñanos motivates regulation of transposase activity to prevent excessive and uncontrolled transposition, they do not disclose the instantly claimed methods of regulating transposase activity.
This deficiency is cured by Strecker. Strecker discloses a system to deliver a helper plasmid containing TnsB, TnsC, TniQ, Cas12k, a tracrRNA, and a synthetic crRNA; a donor plasmid containing DNA flanked by transposition machinery sites; and a target plasmid library (page 207, column 1, paragraph 1). The system of Strecker first supplies the purified proteins disclosed therein (see Figure 5 for a schematic of protein partners involved in RNA-guided DNA transposition) with a buffer comprising EDTA, which is a metal ion chelation agent per the instant specification (page 35, lines 19-21). Following assembling the reaction under these inactivated transposase conditions, Strecker discloses in vitro transposition reactions, wherein the in vitro transposition reaction buffer comprises MgCl2, which is a divalent ion (see section “In vitro Transposition Assays” from supplemental methods). Thus, the disclosure of Strecker, in combination with Cadiñanos, motivates regulation of transposase activity by modifying reaction conditions to restrict transposase activity to acting on the polynucleotide to be characterized using nanopore sequencing, as disclosed in Oxford.
As previously set forth, Oxford discloses a method for modifying a template double stranded polynucleotide, especially for characterization using nanopore sequencing (abstract), said method comprising contacting a template polynucleotide with a MuA transposase under conditions which allow the transposase to function, as the transposition reaction itself requires an activated transposase (page 11, lines 1-8).
Additionally, regarding the removal of any transposase unbound to the polynucleotide-guided effector protein, as set forth above, Cadiñanos, Strecker, and Oxford collectively motivate regulation of transposase activity, as unregulated, active transposases are known to result in genomic instability such as inversions, deletions, and translocations (Cadiñanos: page 1, column 2, paragraph 3). Therefore, it is considered that one of ordinary skill in the art would be aware that binding said transposase to said polynucleotide-guided effector protein constitutes a method of regulating transposase activity, as bound transposases will necessarily be regulated by restricting them to acting on genomic sequences targeted by the polynucleotide-guided effector protein, as in the methodology disclosed in Chavez (and Strecker). Conversely, unbound transposases will necessarily be unregulated and unrestricted from acting on genomic sequences, potentially resulting in the undesired genomic instability disclosed in Cadiñanos. Accordingly, it is considered that these disclosures also motivate removal of unbound transposases to regulate and restrict their activity to user-specified sequences rather than unrestrained transposition. This is supported by the disclosure of Carter, which discloses the antibody-guided chromatin tagmentation method (ACT-seq), wherein Tn5 transposase activity is specifically controlled to construct single-cell libraries for mapping epigenetic marks in single cells (abstract). Per Carter, effective tagmentation includes removal of unbound transposases (see section “Bulk-cell chromatin binding and tagmention” of the methods). Accordingly, it is considered that based on the state of the art at the time of filing, one of ordinary skill in the art would have been motivated to remove unbound transposases to regulate and restrict their activity to user-specified sequences (as disclosed in Chavez and Strecker) rather than the unrestrained transposition disclosed in Cadiñanos for purposes of generating a nucleic acid construct for single molecule characterization (as in Oxford and Carter).
Regarding the insertion of a modified polynucleotide within a transposable element, as previously set forth, Ammar discloses that transposons (which read on the instantly claimed transposable elements) can be co-opted to insert sequences of interest (such as a fluorescent marker) by flanking said sequences with inverted terminal repeat sequences (abstract). The instant specification defines the claimed modified polynucleotide to be “an element that facilitates or improves single molecule characterization, such as a marker, a tether or an adaptor” (page 15, lines 13-15). Thus, the disclosure of Ammar establishes that transposable elements are known to be useful in inserting sequences of interest, such as fluorescent markers (which read on the instantly claimed modified polynucleotide), by flanking said sequences as set forth above and then mobilizing them by supplying the transposase (page 230, paragraph 1).
Finally, regarding the applicability of these methods to single-molecule characterization, as set forth above, Oxford discloses modifying a template double stranded polynucleotide, especially for characterization using nanopore sequencing (abstract), said method comprising contacting a template polynucleotide with a MuA transposase under conditions which allow the transposase to function (page 11, lines 1-8). Furthermore, as previously set forth, Stoddart discloses that transposon-mediated modifications are known to be useful in generating a template strand for characterization with nanopore sequencing (abstract). For example, MuA transposase is capable of ligating markers to a template double stranded polynucleotide, which can then be characterized by nanopore sequencing (Example 3, pages 57-61). Per the disclosure of Stoddart, nanopore sequencing is capable of characterizing a single polynucleotide strand via the strand sequencing method (page 1, paragraph 5), meaning insertion of markers into target polynucleotides using transposon machinery (as disclosed in both Ammar and Stoddart) yields modified target polynucleotides compatible with nanopore sequencing (as disclosed in Stoddard) to characterize a single polynucleotide strand, which reads on the instantly claimed “single molecule characterization.”
Thus, it is considered that the combination of cited art collectively discloses each and every limitation of instant claim 51.
With regard to claim 73, which recites “the guide polynucleotide [of the method of claim 51] is a guide RNA and the polynucleotide-guided effector protein is an RNA-guided effector protein,” as previously set forth, Chavez discloses that the site-programmable transposition system set forth above comprises a helper plasmid containing TnsB, TnsC, TniQ, Cas12k, a tracrRNA, and a synthetic crRNA (page 207, column 1, paragraph 1). The instant specification defines the guide polynucleotide to comprise a crRNA and a tracrRNA (page 19, line 17), meaning the tracrRNA and crRNA disclosed in Chavez read on the instantly claimed guide RNA. Additionally, the Cas12k disclosed in Chavez reads on the instantly claimed RNA-guided endonuclease. Thus, it is considered that Chavez discloses each and every additional limitation of instant claim 73.
With regard to claims 74-76, which respectively recite “the polynucleotide-guided effector protein [of the method of claim 51] is an assembly of multiple protein parts,” that “the polynucleotide-guided effector protein [of the method of claim 74] comprises Cascade, which comprises an assembly of Cas6-Cas7-Cas8 proteins,” and that “the polynucleotide-guided effector protein [of the method of claim 51] is Cas12k,” as previously set forth, Chavez summarizes advances in site-programmable transposition (reported in Klompe and Strecker), in which the system of Klompe comprises Cas6-Cas7-Cas8 (i.e. Cascade, which is an assembly of multiple protein parts) (page 206, column 2, paragraph 3; page 207, column 3, paragraph 1) and the system of Strecker comprises Cas12k (page 207, column 1, paragraph 1). Therefore, the polynucleotide-guided effector proteins disclosed in Chavez read on both the instantly claimed multi-protein complex Cascade and Cas12k. Thus, it is considered that Chavez discloses each and every additional limitation of instant claims 74-76.
With regard to claim 78, which recites “the modified polynucleotide [of the method of claim 51] comprises a click reactive group, a fluorophore, a conjugation agent, a pull down group, a tethering moiety, a marker, a modified base, an abasic residue, and/or a spacer,” as set forth above, Ammar discloses that transposons (which read on the instantly claimed transposable elements) can be co-opted to insert sequences of interest (such as a fluorescent marker) by flanking said sequences with inverted terminal repeat sequences and supplying the transposase (abstract; page 230, paragraph 1). The fluorescent marker of Ammar is considered to read on the instantly claimed fluorophore of claim 78. Thus, it is considered that Ammar discloses each and every additional limitation of instant claim 78.
With regard to claim 79, which recites “the transposable element [of claim 51] is a sequencing adaptor, an amplification adaptor, a hairpin adaptor, a unique molecular identifier, or a rolling circle amplification template,” as set forth above, Oxford discloses modifying a template double stranded polynucleotide, especially for characterization using nanopore sequencing (abstract), said method comprising contacting a template polynucleotide with a MuA transposase under conditions which allow the transposase to function (page 11, lines 1-8). Oxford further discloses that the system taught therein may be used to selectively label polynucleotides targeted for downstream sequencing with a labelling entity that enables sample identification or barcoding (page 7, lines 20-23). In the absence of an explicit definition set forth in the instant specification, it is considered that a labelling entity that enables sample identification or barcoding reads on the instantly claimed unique molecular identifier. Thus, it is considered that Oxford discloses each and every additional limitation of instant claim 79.
With regard to claims 80 and 81, which respectively recite “a method of detecting and/or characterising a target polynucleotide in a sample, comprising:
preparing a nucleic acid construct for single molecule characterisation according to the method of claim 51;
contacting the nucleic acid construct with a membrane comprising a transmembrane pore;
applying a potential difference across the membrane; and
taking one or more measurements resulting from the contacting of the nucleic acid construct with the pore to determine the presence or absence of the target polynucleotide and/or one or more characteristics of the target polynucleotide; thereby detecting and/or characterising the target polynucleotide,”
said characteristics being “selected from
the length of the polynucleotide,
the identity of the polynucleotide,
the sequence of the polynucleotide,
the secondary structure of the polynucleotide and
whether or not the polynucleotide is modified,”
as set forth above, Chavez, Klompe, Strecker, Cadiñanos, Oxford, Carter, Ammar, and Stoddard collectively disclose the method of instant claim 51. Additionally, Stoddart discloses characterization of a polynucleotide modified by MuA transposase and its substrates (page 3, paragraph 2) by “contacting the modified polynucleotide with a transmembrane pore such that at least one strand of the polynucleotide moves through the pore and…taking one or more measurements as the at least one strand moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the at least one strand…thereby characterising the modified polynucleotide” (page 30, paragraph 2). This movement through the transmembrane pore is driven by applying a potential across the pore (page 30, paragraph 3), as in instant claim 80, and is disclosed to characterize the modified polynucleotide at least by determining its sequence (i.e. “strand sequencing”), as in instant claim 81 (page 30, paragraph 3). The disclosure of Stoddart thus establishes that transmembrane pores are known to be capable of characterizing the sequence of target polynucleotides modified with transposases such as the MuA transposase and its substrates (page 3, paragraph 2). Thus, it is considered that the combination of cited art collectively discloses each and every limitation of instant claims 80 and 81.
Given that Chavez discloses site-programmable transposition driven by delivering a polynucleotide-guided effector protein (i.e. Cascade or Cas12k), a guide polynucleotide (i.e. tracrRNA and/or crRNA), transposase (i.e. TnsB), and transposable element (donor plasmid containing DNA flanked by transposition machinery sites) to a target polynucleotide (the target plasmid library), in which the polynucleotide-guided effector protein must complex with the transposon machinery to function (as also disclosed in Klompe and Strecker); that Cadiñanos discloses an inducible piggyBac transposon system to prevent unrestricted and unregulated transposase activity (i.e. via the buffers disclosed in Strecker); that Oxford discloses a method for characterizing a template double stranded polynucleotide using nanopore sequencing comprising contacting a template polynucleotide with a MuA transposase under conditions which allow the transposase to function (i.e. under buffer conditions with the instantly claimed salt concentration); and furthermore that Ammar and Stoddart respectively disclose transposon-mediated insertion of markers and the utility of transposon-mediated insertion markers in characterization by nanopore sequencing, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to modify the system disclosed in Chavez to control transposase activity to prevent unintended transposition (i.e. by inactivating and reactivating said transposase by varying salt concentration and ensuring said transposase is bound to the polynucleotide-guided effector protein, as disclosed in Cadiñanos, Oxford, and Carter), as well as to insert markers (such as the fluorescent markers disclosed in Ammar) to predictably generate a nucleic acid construct for single molecule characterization (such as by nanopore sequencing; as disclosed in Stoddart and Oxford). One would have been motivated to make such a modification in order to receive the expected benefit of harnessing the efficiency and specificity of polynucleotide-guided effector proteins (such as Cas12k) in delivering user-specified modifications such as markers carried by transposons to targeted polynucleotides in a regulated and controlled workflow to facilitate their individual characterization via downstream nanopore sequencing.
Claim 52 is rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (hereinafter Chavez; of record), in view of Cadiñanos and Bradley, 2007 (hereinafter Cadiñanos; of record), Strecker et al., 2019 (hereinafter Strecker; as cited in the IDS filed 07/07/2022), Klompe et al., 2019 (hereinafter Klompe; as cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford; of record), Carter et al., 2019 (hereinafter Carter), Ammar et al., 2012 (hereinafter Ammar; of record), and WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), as applied to claim 51 above, and further in view of Nesmelova and Hackett, 2010 (hereinafter Nesmelova; of record) and Zhang et al., 2019 (hereinafter Zhang; of record).
The combined disclosures of Chavez, Cadiñanos, Strecker, Klompe, Oxford, Carter, Ammar, and Stoddart are described above and applied as before. However, these disclosures do not teach the protein-protein interaction binding the polynucleotide-guided effector protein to the transposase, as in instant claim 52.
With regard to claim 52, which recites “the polynucleotide-guided effector protein [of claim 51] binds to the transposase via a protein-protein interaction,” Nesmelova discloses that the C-terminal domain of the Tn5 transposase is also known as the dimerization domain and is responsible for protein-protein interactions involving the transposase (Figure 1). Additionally, Zhang discloses that polynucleotide-guided effector proteins (such as Cas9 and Cas12a) participate in direct physical interactions with proteins (i.e. inhibitor proteins or anti-CRISPR proteins) (summary; page 816, column 1, paragraph 3), which read on the instantly claimed protein-protein interaction. As set forth above regarding instant claim 51, Chavez discloses Cas12k must associate with TniQ, TnsB, and TnsC (the Cascade-TniQ complex) in order to function properly. Thus, it was known prior to the effective filing date of the invention not only that the polynucleotide-guided effector protein must physically associate with a transposase-containing complex, but also that both Tn5 (i.e. a transposase) and Cas endonucleases such as Cas9 and Cas12a (i.e. polynucleotide-guided effector proteins) are each capable of protein-protein interactions.
Given that Chavez, Cadiñanos, Strecker, Klompe, Oxford, Carter, Ammar, and Stoddart collectively disclose the method of instant claim 51 (as set forth above), with Chavez specifically disclosing that the polynucleotide-guided effector protein must complex with the transposon machinery to function (as set forth above); that Nesmelova discloses that the C-terminal domain of the Tn5 transposase governs protein-protein interactions involving the transposase; and that Zhang discloses that polynucleotide-guided effector proteins such as Cas9 and Cas12a also participate in protein-protein interactions, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to form a complex between the instantly claimed polynucleotide-guided effector protein and transposase via protein-protein interactions to predictably produce an active complex capable of targeted transposon-mediated marker insertion (as set forth above regarding instant claim 51). One would have been motivated to make such a modification in order to receive the expected benefit of producing an active complex capable of targeted transposon-mediated marker insertion into a target polynucleotide to facilitate its individual characterization via nanopore sequencing.
Claims 53, 54, and 77 are rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (hereinafter Chavez; of record), in view of Cadiñanos and Bradley, 2007 (hereinafter Cadiñanos; of record), Strecker et al., 2019 (hereinafter Strecker; as cited in the IDS filed 07/07/2022), Klompe et al., 2019 (hereinafter Klompe; as cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford; of record), Carter et al., 2019 (hereinafter Carter), Ammar et al., 2012 (hereinafter Ammar; of record), and WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), as applied to claim 51 above, and further in view of Bhatt and Chalmers, 2019 (hereinafter Bhatt; of record), as evidenced by Bouuaert and Chalmers, 2017 (hereinafter Bouuaert; of record).
The combined disclosures of Chavez, Cadiñanos, Strecker, Klompe, Oxford, Carter, Ammar, and Stoddart are described above and applied as before. However, these disclosures do not teach the genetic fusion of the polynucleotide-guided effector protein to the transposase or the linker connecting the same, as in instant claims 53 and 54. Nor do these disclosures teach the specific multimeric transposase of instant claim 77.
With regard to claim 53, which recites “the polynucleotide-guided effector protein [of claim 51] is genetically fused to the transposase,” Bhatt discloses that Cas9 may be used to achieve targeted integration via the mariner-family transposon Hsmar1 (abstract). They further disclose that this may be accomplished by fusing dCas9 to a transposase (Figures 3 and 4), which does not interfere with transposase activity and is thus a promising system for targeted integration (page 8133, column 2, paragraph 3). The dCas9-transposase fusion of Bhatt thus reads on the instantly claimed genetic fusion of the polynucleotide-guided effector protein and the transposase.
With regard to claim 54, which recites “the polynucleotide-guided effector protein [of claim 51] is connected to the transposase via a linker moiety,” while Bhatt reports the success of targeted integration driven by fusing dCas9 to a transposase (Figures 3 and 4), they also disclose that linkers connecting polynucleotide-guided effector proteins such as zinc fingers or TALE proteins to transposases such as piggyBac have been previously reported to function in targeted integration (page 8133, column 2, paragraph 2). The linker connecting polynucleotide-guided effector proteins such as zinc fingers or TALE proteins to transposases such as piggyBac disclosed in Bhatt thus reads on the instantly claimed linker connecting the polynucleotide-guided effector protein and the transposase.
With regard to claim 77, which recites “the transposase [of claim 51] is a multimeric protein and the multimeric protein comprises the maize Ac transposon, the Drosophila P element, Tn5, Tn7, Tn10, Mariner, IS10, IS50 or MuA,” Bhatt discloses that Cas9 may be used to achieve targeted integration by fusing dCas9 to the mariner-family transposon Hsmar1 (abstract; Figures 3 and 4), as set forth above. Per the teachings of Bouuaert, the transposase acting with Hsmar1 is a dimer (Figure 1), meaning the system disclosed in Bhatt comprises both a multimeric transposase and a Mariner transposon, as instantly claimed.
Given that Chavez, Cadiñanos, Strecker, Klompe, Oxford, Carter, Ammar, and Stoddart collectively disclose the method of instant claim 51 (as set forth above), with Chavez specifically disclosing that the polynucleotide-guided effector protein must complex with the transposon machinery to function (as set forth above), and that Bhatt discloses both genetic fusion of a polynucleotide-guided effector protein (dCas9) to a multimeric transposase (acting on mariner transposon Hsmar1; as disclosed in Bouuaert) and connection of the same via a linker, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to form a complex between the instantly claimed polynucleotide-guided effector protein and multimeric transposase acting on mariner transposon Hsmar1 via genetic fusion or a linker to predictably produce an active complex capable of targeted transposon-mediated marker insertion (as set forth above regarding instant claim 51). One would have been motivated to make such a modification in order to receive the expected benefit of producing an active complex capable of targeted Hsmar1 transposon-mediated marker insertion into a target polynucleotide (via the action of a multimeric transposase) to facilitate its individual characterization via nanopore sequencing.
Claims 71 and 72 are rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (hereinafter Chavez; of record), in view of Cadiñanos and Bradley, 2007 (hereinafter Cadiñanos; of record), Strecker et al., 2019 (hereinafter Strecker; as cited in the IDS filed 07/07/2022), Klompe et al., 2019 (hereinafter Klompe; as cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford; of record), Carter et al., 2019 (hereinafter Carter), Ammar et al., 2012 (hereinafter Ammar; of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), and Bhatt and Chalmers, 2019 (hereinafter Bhatt; of record), as evidenced by Bouuaert and Chalmers, 2017 (hereinafter Bouuaert; of record), as applied to claim 54 above, and further in view of WO 2019/070762 (hereinafter Lee; of record), as evidenced by Sakamoto et al., 2017 (hereinafter Sakamoto; of record) and Jiang and Doudna, 2017 (hereinafter Jiang; of record).
The combined disclosures of Chavez, Cadiñanos, Strecker, Klompe, Oxford, Carter, Ammar, Stoddart, Chatt, and Bouuaert, are described above and applied as before. However, these disclosures do not teach the linker moiety limitations of instant claims 71 and 72.
With regard to claims 71 and 72, which respectively recite “the linker moiety [of claim 54] is a linker polynucleotide,” “wherein the linker moiety sequence determines the length of the sequence between a protospacer adjacent motif (PAM) immediately upstream of the sequence within the target polynucleotide contacted by the polynucleotide-guided effector protein and the site in which the transposase inserts the transposable element into the region of interest,” Lee discloses crRNA sequences modified with an extension sequence (paragraphs [0049] and [0050]) that has another function, such as being an aptamer (paragraph [0054]) in addition to binding to a polynucleotide-guided effector protein (Cpf1) (paragraph [0046]). Per Sakamoto, aptamers are nucleic acids that bind to a broad range of target molecules including proteins (page 91, column 1, paragraph 1). Under broadest reasonable interpretation, proteins encompass enzymes such as the instantly claimed transposase, meaning the crRNA extension aptamers of Lee are nucleic acids considered to be capable of linking the instantly claimed transposase and the instantly claimed polynucleotide-guided effector protein, as in instant claim 71.
Additionally, the recited limitations regarding the linker moiety sequence determining the length of the sequence between a PAM and the targeted site (see instant claim 72) are considered to be inherent properties conferred by crRNA design, as evidenced by Jiang. Per Jiang, crRNA directs the polynucleotide-guided effector protein (i.e. a Cas endonuclease) to the cleavage target site, which is defined and identified by its complementarity to the crRNA sequence preceding the PAM sequence (page 506, Figure 1 caption).
Given that Chavez, Cadiñanos, Strecker, Klompe, Oxford, Carter, Ammar, and Stoddart collectively disclose the method of instant claim 51 (as set forth above), with Chavez specifically disclosing that the polynucleotide-guided effector protein must complex with the transposon machinery to function (as set forth above), and that Lee discloses a crRNA capable of binding to a polynucleotide-guided effector protein and further modified by an extension capable of functioning as an aptamer, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to link the polynucleotide-guided effector (bound by a crRNA) to the transposase via the crRNA extension functioning as an aptamer to predictably produce an active complex capable of targeted transposon-mediated marker insertion (as set forth above regarding instant claim 51) in which target specificity is conferred by crRNA design (as evidenced by Jiang). One would have been motivated to make such a modification in order to receive the expected benefit of producing an active complex capable of targeted transposon-mediated marker insertion into a target polynucleotide to facilitate its individual characterization downstream.
Claims 82-86 are rejected under 35 U.S.C. 103 as being unpatentable over Chavez and Qi, 2019 (hereinafter Chavez; of record), in view of Cadiñanos and Bradley, 2007 (hereinafter Cadiñanos; of record), Strecker et al., 2019 (hereinafter Strecker; as cited in the IDS filed 07/07/2022), Klompe et al., 2019 (hereinafter Klompe; as cited in the IDS filed 07/07/2022), WO 2017/203267 A1 (hereinafter Oxford; of record), Carter et al., 2019 (hereinafter Carter), Ammar et al., 2012 (hereinafter Ammar; of record), WO 2015/022544 (hereinafter Stoddart; of record; cited in the IDS filed 07/07/2022), Bhatt and Chalmers, 2019 (hereinafter Bhatt; of record), and WO 2019/070762 (hereinafter Lee; of record), as evidenced by Sakamoto et al., 2017 (hereinafter Sakamoto; of record)
With regard to claim 82, which recites “a method of preparing a nucleic acid construct, comprising, in a reaction vessel:
(a) inactivating a transposase by:…
(ii) providing a metal ion chelation agent,…
(b) contacting a target polynucleotide with:
a polynucleotide-guided effector protein,
a guide polynucleotide,
the inactivated transposase, and
a transposable element;
wherein the polynucleotide-guided effector protein and transposase are genetically fused or connected via a linker moiety; and
wherein the inactivated transposase is directed to a region of interest within the target polynucleotide; and
(d) reactivating the inactivated transposase by:…
(iii) providing a solution comprising divalent ions…wherein the reactivated transposase inserts the transposable element into the target polynucleotide; thereby preparing a nucleic acid construct,”
as set forth above regarding instant claim 51, Chavez summarizes advances in site-programmable transposition (reported in Klompe and Strecker, both of which are cited in the IDS filed 07/07/2022), specifically disclosing that Strecker engineered a system to deliver a helper plasmid containing TnsB, TnsC, TniQ, Cas12k, a tracrRNA, and a synthetic crRNA; a donor plasmid containing DNA flanked by transposition machinery sites; and a target plasmid library (page 207, column 1, paragraph 1). This system thus delivers the instantly claimed polynucleotide-guided effector protein (Cas12k), a guide polynucleotide (tracrRNA and crRNA), transposase (TnsB), and transposable element (donor plasmid containing DNA flanked by transposition machinery sites) to a target polynucleotide (the target plasmid library). However, Chavez does not disclose the inactivation or reactivation of said transposase, nor do they disclose the linker moiety connecting the polynucleotide-guided effector protein and transposase. However, these deficiencies are cured by various secondary references, as set forth below.
Regarding the inactivation and reactivation of the claimed transposase, as previously set forth, Cadiñanos discloses an inducible piggyBac transposon system (abstract), in which piggyBac activity is regulated by the presence or absence of 4- hydroxytamoxifen (page 4, column 2, paragraph 2-page 5, column 1, paragraph 1; Figure 4). Cadiñanos explicitly discloses that this inducible system was developed to prevent excessive and uncontrolled transposition mediated by active transposases that results in genomic instability such as inversions, deletions, and translocations by facilitating temporal regulation of transposition activity (page 1, column 2, paragraph 3; page 6, column 2, paragraph 1-page 7, column 1, paragraph 1). Thus, while the disclosure of Cadiñanos motivates regulation of transposase activity to prevent excessive and uncontrolled transposition, they do not disclose the instantly claimed methods of regulating transposase activity.
This deficiency is cured by Strecker. As set forth above, Strecker discloses a system to deliver a helper plasmid containing TnsB, TnsC, TniQ, Cas12k, a tracrRNA, and a synthetic crRNA; a donor plasmid containing DNA flanked by transposition machinery sites; and a target plasmid library (page 207, column 1, paragraph 1). The system of Strecker first supplies the purified proteins disclosed therein (see Figure 5 for a schematic of protein partners involved in RNA-guided DNA transposition) with a buffer comprising EDTA, which is a metal ion chelation agent per the instant specification (page 35, lines 19-21). Following assembling the reaction under inactivated transposase conditions, Strecker discloses varying out of in vitro transposition reactions, wherein the in vitro transposition reaction buffer comprises MgCl2, which is a divalent ion (see section “In vitro Transposition Assays” from supplemental methods). Thus, the disclosure of Strecker, in combination with Cadiñanos, motivates regulation of transposase activity by modifying reaction conditions to restrict transposase activity to acting on the polynucleotide to be characterized using nanopore sequencing, as disclosed in Oxford.
As previously set forth, Oxford discloses a method for modifying a template double stranded polynucleotide, especially for characterization using nanopore sequencing (abstract), said method comprising contacting a template polynucleotide with a MuA transposase under conditions which allow the transposase to function, as the transposition reaction itself requires an activated transposase (page 11, lines 1-8).
With regard to the linker moiety connecting the polynucleotide-guided effector protein and transposase, as set forth above, while Bhatt reports the success of targeted integration driven by fusing dCas9 to a transposase (Figures 3 and 4), they also disclose that linkers connecting polynucleotide-guided effector proteins such as zinc fingers or TALE proteins to transposases such as piggyBac have been previously reported to function in targeted integration (page 8133, column 2, paragraph 2). The linker connecting polynucleotide-guided effector proteins such as zinc fingers or TALE proteins to transposases such as piggyBac disclosed in Bhatt thus reads on the instantly claimed linker connecting the polynucleotide-guided effector protein and the transposase.
Regarding the insertion of a modified polynucleotide within a transposable element, as previously set forth, Ammar discloses that transposons (which read on the instantly claimed transposable elements) can be co-opted to insert sequences of interest (such as a fluorescent marker) by flanking said sequences with inverted terminal repeat sequences (abstract). The instant specification defines the claimed modified polynucleotide to be “an element that facilitates or improves single molecule characterization, such as a marker, a tether or an adaptor” (page 15, lines 13-15). Thus, the disclosure of Ammar establishes that transposable elements are known to be useful in inserting sequences of interest, such as fluorescent markers (which read on the instantly claimed modified polynucleotide), by flanking said sequences as set forth above and then mobilizing them by supplying the transposase (page 230, paragraph 1).
Finally, regarding the applicability of these methods to single-molecule characterization, as set forth above, Oxford discloses modifying a template double stranded polynucleotide, especially for characterization using nanopore sequencing (abstract), said method comprising contacting a template polynucleotide with a MuA transposase under conditions which allow the transposase to function (page 11, lines 1-8). Furthermore, as previously set forth, Stoddart discloses that transposon-mediated modifications are known to be useful in generating a template strand for characterization with nanopore sequencing (abstract). For example, MuA transposase is capable of ligating markers to a template double stranded polynucleotide, which can then be characterized by nanopore sequencing (Example 3, pages 57-61). Per the disclosure of Stoddart, nanopore sequencing is capable of characterizing a single polynucleotide strand via the strand sequencing method (page 1, paragraph 5), meaning insertion of markers into target polynucleotides using transposon machinery (as disclosed in both Ammar and Stoddart) yields modified target polynucleotides compatible with nanopore sequencing (as disclosed in Stoddard) to characterize a single polynucleotide strand, which reads on the instantly claimed “single molecule characterization.”
Thus, it is considered that the combination of cited art collectively discloses each and every limitation of instant claim 82.
With regard to claim 83, which recites “the transposable element [of the method of claim 82] comprises a modified polynucleotide,” as set forth above, Ammar discloses that transposons (which read on the instantly claimed transposable elements) can be co-opted to insert sequences of interest (such as a fluorescent marker) by flanking said sequences with inverted terminal repeat sequences (abstract). The instant specification defines the claimed modified polynucleotide to be “an element that facilitates or improves single molecule characterization, such as a marker, a tether or an adaptor” (page 15, lines 13-15). Thus, the disclosure of Ammar establishes that transposable elements are known to be useful in inserting sequences of interest, such as fluorescent markers (which read on the instantly claimed modified polynucleotide), by flanking said sequences as set forth above and then mobilizing them by supplying the transposase (page 230, paragraph 1). Thus, it is considered that Ammar discloses each and every additional limitation of instant claim 83.
With regard to claim 84, which recites “the linker moiety [of the method of claim 82] is a linker polynucleotide that binds to the polynucleotide-guided effector protein and to the transposase,” as set forth above, Lee discloses crRNA sequences modified with an extension sequence (paragraphs [0049] and [0050]) that has another function, such as being an aptamer (paragraph [0054]) in addition to binding to a polynucleotide-guided effector protein (Cpf1) (paragraph [0046]). Per Sakamoto, aptamers are nucleic acids that bind to a broad range of target molecules including proteins (page 91, column 1, paragraph 1). Under broadest reasonable interpretation, proteins encompass enzymes such as the instantly claimed transposase, meaning the crRNA extension aptamers of Lee are nucleic acids considered to be capable of linking the instantly claimed transposase and the instantly claimed polynucleotide-guided effector protein, as in instant claim 84. Thus, it is considered that Lee and Sakamoto collectively disclose each and every additional limitation of instant claim 84.
With regard to claim 85, which recites “the contacting of (b) [of the method of claim 82] comprises contacting the target polynucleotide with a complex comprising the polynucleotide-guided effector protein, the guide polynucleotide, the transposase, and the transposable element,” as set forth above, Chavez discloses that the Cas12k taught therein must associate with TniQ, TnsB, and TnsC in order for this system to function properly (page 207, column 3, paragraph 1; Figure 1). This association reads on the instantly claimed “complex.” Furthermore, as is illustrated in Figure 1, this system only functions when all necessary components associate together, including the donor DNA, which is considered to comprise the instantly claimed transposable element, as set forth above. Thus, Chavez discloses a complex comprising a polynucleotide-guided effector protein guided to its target site by its bound guide polynucleotide, a transposase, and a transposable element, as instantly claimed. Thus, Chavez discloses each and every additional limitation of instant claim 85.
With regard to claim 86, which recites “a method of detecting and/or characterising a target polynucleotide in a sample, comprising:
preparing a nucleic acid construct according to the method of claim 82;
contacting the nucleic acid construct with a membrane comprising a transmembrane pore;
applying a potential difference across the membrane; and
taking one or more measurements resulting from the contacting of the nucleic acid construct with the pore to determine the presence or absence of the target polynucleotide and/or one or more characteristics of the target polynucleotide; thereby detecting and/or characterising the target polynucleotide,”
Chavez, Klompe, Strecker, Cadiñanos, Oxford, Bhatt, and Ammar collectively disclose the method of claim 82, as set forth above. Additionally, Stoddart discloses characterization of a polynucleotide modified by MuA transposase and its substrates (page 3, paragraph 2) by “contacting the modified polynucleotide with a transmembrane pore such that at least one strand of the polynucleotide moves through the pore and…taking one or more measurements as the at least one strand moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the at least one strand…thereby characterising the modified polynucleotide” (page 30, paragraph 2). This movement through the transmembrane pore is driven by applying a potential across the pore (page 30, paragraph 3), as in instant claim 80, and is disclosed to characterize the modified polynucleotide at least by determining its sequence (i.e. “strand sequencing”), as in instant claim 81 (page 30, paragraph 3). The disclosure of Stoddart thus establishes that transmembrane pores are known to be capable of characterizing the sequence of target polynucleotides modified with MuA transposase and its substrates (page 3, paragraph 2). Thus, it is considered that Stoddart discloses each and every additional limitation of instant claim 86.
Given that Chavez discloses site-programmable transposition driven by delivering a polynucleotide-guided effector protein (i.e. Cascade or Cas12k), a guide polynucleotide (i.e. tracrRNA and/or crRNA), transposase (i.e. TnsB), and transposable element (donor plasmid containing DNA flanked by transposition machinery sites) to a target polynucleotide (the target plasmid library), in which the polynucleotide-guided effector protein must complex with the transposon machinery to function (as also disclosed in Klompe and Strecker); that Cadiñanos discloses an inducible piggyBac transposon system to prevent unrestricted and unregulated transposase activity (i.e. via the buffers disclosed in Strecker); that Oxford discloses a method for characterizing a template double stranded polynucleotide using nanopore sequencing comprising contacting a template polynucleotide with a MuA transposase under conditions which allow the transposase to function (i.e. under buffer conditions with the instantly claimed salt concentration); Ammar and Stoddart respectively disclose transposon-mediated insertion of markers and the utility of transposon-mediated insertion markers in characterization by nanopore sequencing and finally that Bhatt discloses both genetic fusion of a polynucleotide-guided effector protein (dCas9) to a multimeric transposase (acting on mariner transposon Hsmar1) and connection of the same via a linker (such as the polynucleotide linker disclosed in Lee), it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to modify the system disclosed in Chavez to ensure functional complex formation by connecting the polynucleotide-guided effector protein and transposase with a linker (as disclosed in Bhatt) such as a polynucleotide linker (as disclosed in Lee), to control transposase activity to prevent unintended transposition (i.e. by inactivating and reactivating said transposase by varying salt concentration, as disclosed in Cadiñanos and Oxford), as well as to insert markers (such as the fluorescent markers disclosed in Ammar) to predictably generate a nucleic acid construct for purposes such as single molecule characterization (i.e. nanopore sequencing) (as disclosed in Oxford and Stoddart). One would have been motivated to make such a modification in order to receive the expected benefit of harnessing the efficiency and specificity of polynucleotide-guided effector proteins (such as Cas12k) in delivering user-specified modifications such as markers carried by transposons to targeted polynucleotides in a regulated and controlled workflow to facilitate their individual characterization via downstream nanopore sequencing.
Conclusion
No claims are allowed.
Claim 82 is objected to.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637
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