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 .
DETAILED ACTION
Status of the Claims
Claims 1, 6-11, 13-15, 18-20, 23, 25, 33-34, 36 and 38-39 are pending. Claims 1, 6-11, 13-15, 25, 34, 36 and 38-39 are the subject of this FINAL Office Action. Claims 18-20, 23 and 33 are withdrawn.
The prior art rejections are based on the Third-Party Submission 07/13/2023, with which the Examiner agrees. This also reflects the European Opposition filed 04/13/2023.
Election/Restrictions
Applicant’s election without traverse of the species of (1) polynucleotide “leader” (2) Dda helicase “polynucleotide-handling protein”, (3) conjugate of claim 15 and (4) adaptor attachment in the reply filed on 09/02/2025 was acknowledged. The elections read on claims 1, 6-11, 13-15, 25, 32 and 34-37.
Claims 18-20, 23 and 33 are withdrawn.
Claim Interpretations
As to a “leader” intended to “facilitate[] the threading of the conjugate through the nanopore,” again the structure required is never defined, but is broadly interpreted as any nucleic acid sequence (see para. 0025- “L is a leader, wherein L is optionally an N moiety . . . N comprises a polynucleotide”), or [charged] polymer (e.g. PEG, polysaccharide, nylon, polyethylene, polypropylene, etc.; see para. 0221- “ In some embodiments the leader comprises a polymer”). Yet, the only specific example of leader PN is disclosed in Example 1: “The Y adapter contains 30 C3 leader section for easier capture by the nanopore and a side arm for tethering to the membrane” (para. 0386). As to “charged polymer,” the only description is
In some embodiments the leader is a charged polymer, e.g. a negatively charged polymer. In some embodiments the leader comprises a polymer such as PEG or a polysaccharide. In such embodiments the leader may be from 10 to 150 monomer units (e.g. ethylene glycol or saccharide units) in length, such as from 20 to 120, e.g. 30 to 100, for example 40 to 80 such as 50 to 70 monomer units (e.g. ethylene glycol or saccharide units) in length
(spec., para. 0221). This is, once again, any polymer with any amount of charge.
As to claims 6-8, the claimed functionality (“capable of”) of the “polynucleotide-handling protein” is never clearly tied to any particular structure in the specification; thus, claims 6-8 encompass any “polynucleotide-handling protein.” For example, the specification discloses that helicases perform these functions; thus, any prior art helicase meets the claimed functions.
As to “adaptors,” “tethers” and “anchors,” again no definition is provided; they encompass any polynucleotides.
Claim 25 merely recites an intended use of the measurements; thus, it does not further limit the method of claim 1.
As should be clear from the above comments, the claims are much too broad to warrant allowable subject matter. Significant amendments are required.
Claim Rejections - 35 USC § 112- Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(B) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 6-8 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
This same analysis applies to claim 6-8, where the claims also state pure functionality of the claimed “polynucleotide-handling protein.” For example, claim 6 states “the polynucleotide-handling protein is capable of remaining bound to the conjugate when the portion of the conjugate in contact with the active site of the polynucleotide-handling protein comprises a polypeptide.” The specification simply never explains what specific structures yield this result. A skilled artisan is left to guess what among thousands of potential “proteins” allows these particular functions in this nanopore context.
Response to Arguments
The rejection of claims 6-8 is maintained because they fail to clearly and unambiguously point to specific structural features of topoisomerases, exonucleases, helicases or polymerases that are clearly correlated with the claimed functions. First, the range of “polynucleotide-handling proteins” is wide. The claims allows for any of topoisomerases, exonucleases, helicases or polymerases. Within each of these classes (each with different structure and correlative functions) is a wide range of species. For example, polymerases encompasses Taq, Phi29, Bst, various RNA polymerases, TdT, Poly-A polymerase, Polβ, etc. Each has different general structure and correlative functions. Yet, the specification never clearly correlates any particular structural features of each of the above classes or species with any particular claimed function. Again, the claimed functions are: “capable of remaining bound to the conjugate when the portion of the conjugate in contact with the active site of the polynucleotide-handling protein comprises a polypeptide” (claim 6); “modified to prevent it from disengaging from the conjugate when the polynucleotide-handling protein contacts a portion of the conjugate comprising a polypeptide” (claim 7); and “modified to wholly or partially close an opening existing in at least one conformation state of the unmodified protein through which a polynucleotide strand can unbind” (claim 8). The specification only discloses a wish or a plan to further research to discover the possible structural features of the claimed “polynucleotide-handling proteins” that cause these functions.
In some embodiments the polynucleotide-handling protein is capable of remaining bound to the conjugate when the portion of the conjugate in contact with the active site of the polynucleotide-handling protein comprises a polypeptide. In other words, in some embodiments the polynucleotide-handling protein does not dissociate from the conjugate when the polynucleotide-handling protein contacts the polypeptide portion of the conjugate. In some embodiments the polynucleotide-handling protein moves freely with respect to the polypeptide portion until one or more subsequent polynucleotide portions of the conjugate are contacted.
In some embodiments the polynucleotide-handling protein is modified to prevent it from disengaging from the conjugate, polynucleotide or adapter (other than by passing off the end of the conjugate, polynucleotide or adapter) when the polynucleotide-handling protein contacts a portion of the conjugate comprising a polypeptide. Such modified polynucleotide-handling proteins are particularly suitable for use in the disclosed methods.
The polynucleotide-handling protein can be adapted in any suitable way. For example, the polynucleotide-handling protein can be loaded onto the polynucleotide, conjugate or adapter and then modified in order to prevent it from disengaging. Alternatively, the polynucleotide-handling protein can be modified to prevent it from disengaging before it is loaded onto the polynucleotide, conjugate or adapter. Modification of a polynucleotide-handling protein in order to prevent it from disengaging from a polynucleotide, conjugate or adapter can be achieved using methods known in the art, such as those discussed in WO 2014/013260, which is hereby incorporated by reference in its entirety, and with particular reference to passages describing the modification of polynucleotide-handling proteins (polynucleotide binding proteins) such as helicases in order to prevent them from disengaging with polynucleotide strands.
For example, the polynucleotide-handling protein may have a polynucleotide-unbinding opening; e.g. a cavity, cleft or void through which a polynucleotide strand may pass when the polynucleotide-handling protein disengages from the strand. In some embodiments, the polynucleotide-unbinding opening for a given motor protein (polynucleotide-handling protein) can be determined by reference to its structure, e.g. by reference to its X-ray crystal structure. The X-ray crystal structure may be obtained in the presence and/or the absence of a polynucleotide substrate. In some embodiments, the location of a polynucleotide-unbinding opening in a given polynucleotide-handling protein may be deduced or confirmed by molecular modelling using standard packages known in the art. In some embodiments, the polynucleotide-unbinding opening may be transiently produced by movement of one or more parts e.g. one or more domains of the polynucleotide-handling protein.
The polynucleotide-handling protein (motor protein) may be modified by closing the polynucleotide-unbinding opening. Closing the polynucleotide-unbinding opening may therefore prevent the polynucleotide-handling protein from disengaging from the polypeptide portion of the conjugate as well as preventing it from disengaging from the polynucleotide or adapter. For example, the motor protein may be modified by covalently closing the polynucleotide-unbinding opening. In some embodiments, a motor protein for addressing in this way is a helicase, as described herein. Accordingly, in some embodiments of the disclosed methods, the polynucleotide-handling protein is modified to wholly or partially close an opening existing in at least one conformation state of the unmodified protein through which a polynucleotide strand can unbind
(paras. 0276-80). Applicants have failed to describe any structures here. On the contrary, the claimed functions are described as discoverable using known techniques such as x-ray crystallography (“In some embodiments, the polynucleotide-unbinding opening for a given motor protein (polynucleotide-handling protein) can be determined by reference to its structure, e.g. by reference to its X-ray crystal structure. The X-ray crystal structure may be obtained in the presence and/or the absence of a polynucleotide substrate. In some embodiments, the location of a polynucleotide-unbinding opening in a given polynucleotide-handling protein may be deduced or confirmed by molecular modelling using standard packages known in the art.”). At best, paragraph 0278 states
Modification of a polynucleotide-handling protein in order to prevent it from disengaging from a polynucleotide, conjugate or adapter can be achieved using methods known in the art, such as those discussed in WO 2014/013260, which is hereby incorporated by reference in its entirety, and with particular reference to passages describing the modification of polynucleotide-handling proteins (polynucleotide binding proteins) such as helicases in order to prevent them from disengaging with polynucleotide strands.
However, incorporation-by-reference of essential subject matter requires reference to United States patent or patent publication. See 37 C.F.R. § 1.57(d) (“(d) "Essential material" may be incorporated by reference, but only by way of an incorporation by reference to a U.S. patent or U.S. patent application publication, which patent or patent application publication does not itself incorporate such essential material by reference.”). WO 2014/013260 is not a U.S. patent or U.S. patent application publication. Thus, paragraph 0278 fails to disclose any structures.
Even if this deficiency was corrected (see MPEP § 608.01(p)(A)(2)), yet WO 2014/013260 only discloses “the size of the opening [of a helicase] is reduced or the opening is closed by connecting at least two parts of the helicase.” “In particular, the inventors have surprisingly demonstrated that helicases modified in accordance with the invention will strongly bind to a long polynucleotide, such as a polynucleotide comprising 400 nucleotides or more, and will control the movement of most of, if not all of, the polynucleotide.” The helicase structure disclosed is entitled “Hel308.” It contains “one or more cysteine residues and/or one or more non- natural amino acids [that] have been introduced at one or more of the positions which correspond to D272, N273, D274, G281, E284, E285, E287, S288, T289, G290, E291, D293, T294, N300, R303, K304, N314, S315, N316, H317, R318, K319, L320, E322, R326, N328, S615, K717, Y720, N721 and S724 in Hel308 Mbu (SEQ ID NO: 10).” There is nothing about polymerases, exonucleases, or topoisomerases. And the helicase structures disclosed are specific to those of Hel308. No other helicase is disclosed, much less for anything less than a 400 bp polynucleotide. Thus, at the very most, assuming the improper incorporation-by-reference is corrected, the specification still only discloses Hel308, which matches a very specific type of polynucleotide-binding of claim 8 (claims 7-8 are polypeptide-binding).
In sum, claims 6-8 are indefinite because the structures, other than possibly Hel308 on a 400+ bp polynucleotide, required by the claims are unclear.
Claim Rejections - 35 USC § 102-Maintained-in-Part
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 –
(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, 6-9, 11, 13, 25, 34, 36 and 38-39 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by BAI (US20220365094).
As to claim 1, BAI teaches characterizing polypeptide (pp) (para. 0020) by conjugating pp to polynucleotide (pn) (para. 0021); contacting the conjugate with a polynucleotide-handling protein capable of controlling the movement of the polynucleotide with respect to a nanopore (paras. 0022-23); and taking one or more measurements characteristic of the polypeptide as the conjugate moves with respect to the nanopore, thereby characterising the polypeptide (para. 0024), wherein the conjugate comprises a leader that facilitates the threading of the conjugate through the nanopore (para. 0085 and Fig. 2). As to claim 1, BAI teaches leader pn or polymer (para. 0084 and Fig. 1).
As to claims 6-9, BAI teaches helicase (para. 0006).
As to claim 11, BAI teaches polypeptide 2-50 amino acids (para. 0098).
As to claim 13, BAI teaches polynucleotide 10-1000 nucleotides (id.)
As to claim 25, BAI teaches the one or more measurements are characteristic of one or more characteristics of the polypeptide selected from (i) the length of the polypeptide, (ii) the identity of the polypeptide, (iii) the sequence of the polypeptide, (iv) the secondary structure of the polypeptide and (v) whether or not the polypeptide is modified (para. 0007).
As to claim 34, BAI teaches ion flow current measured (para. 0020).
As to claims 36 and 38, BAI teaches pore protein is MspA (para. 0019).
Response to Arguments
Applicants’ arguments as to BAI are not convincing because the claims do not require two polynucleotides conjugated to a polypeptide. Instead, the claims only require one polynucleotide, which can also contain the “leader.” This is clear from the broad structure of the claims. Claim 1 states “conjugating the target polypeptide to a polynucleotide to form a polynucleotide-polypeptide conjugate,” and “wherein the conjugate further comprises a leader that facilitates the threading of the conjugate through the transmembrane protein.” Nothing in claim 1 requires a second polynucleotide.
BAI teaches, in Figure 2, a polynucleotide conjugated to a polypeptide, the polynucleotide functioning as a leader as claimed, with the same generic polynucleotide structure.
FIG. 2 illustrates the process of the ssDNA-polypeptide conjugate passing through a nanopore in the presence of a polynucleotide binding enzyme: the polynucleotide-polypeptide conjugate quickly passes through the nanopore, and in the action of the polynucleotide binding enzyme, the polynucleotide pulls the conjugated polypeptide to pass through the nanopore again at a controlled speed, and when the polypeptide passes through the nanopore, the polypeptide is determined
(para. 0085; emphases added). Figure 2 shows:
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154
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The curved bottom portion shows the claimed “leader.” The claims remain anticipated.
As to claim 39, BAI clearly teaches CsgG nanopore (e.g. para. 0077).
New Grounds of 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.
Claim(s) 1, 6-11, 13-15, 25, 34, 36 and 38-39 is/are rejected under 35 U.S.C. 103 as being unpatentable over BAI, in view of BAYLEY (WO2015040423), in view of HERON (WO2014013260) and JAYASINGHE (WO2017149317).
The prior art as a whole demonstrates that it would have been obvious to a skilled artisan at the time of filing to apply familiar nanopore modulating techniques to the nanopore modulation of BAI and BAYLEY with a reasonable expectation of success.
As explained above, BAI and BAYLEY teach to measure and detect polypeptide sequences and structures in nanopores using modulated movement in the nanopore. Their methods include the use of helicase to do so.
As to claim 1, BAYLEY teaches characterizing polypeptide (pp) (pg. 7, ll. 18-25) by conjugating pp to polynucleotide (pn) (pg. 10, ll. 31-33); contacting the conjugate with a polynucleotide-handling protein capable of controlling the movement of the polynucleotide with respect to a nanopore (pg. 10, ll. 29-31); and taking one or more measurements characteristic of the polypeptide as the conjugate moves with respect to the nanopore, thereby characterising the polypeptide (pg. 7, ll. 22-24), wherein the conjugate comprises a leader that facilitates the threading of the conjugate through the nanopore (pg. 10, ll. 28-29).
As to claim 10, BAYLEY teaches plurality of and/or polynucleotide (pg. 10, ll. 26-29).
As to claim 32, BAYLEY teaches leader pn or polymer (pg. 10, ll. 28-29; pg. 35, ll. 4-8; Fig. 1).
As to claim 34, BAYLEY teaches ion flow current measured (pg. 7, ll. 22-24; pg. 32, ll. 15-18).
BAYEY does not disclose wherein the polynucleotide-handling protein is a helicase, a polymerase, an exonuclease, or a topoisomerase. Instead, it is an unfoldase.
Neither BAI nor BAYLEY explicitly teach adaptors, tethers and/or anchors (claim 14); or cis-trans configuration of helicase and conjugate (claim 15).
However, these were known techniques to further control movement through a nanopore. For example, HERON teaches helicases such as Dda helicases to perform the functions of claims 6-8, and further control movement through the nanopore (pgs. 1, 18 & 23). To this similar end, JAYASINGHE teaches helicase combined with double-stranded Y-adaptors, anchors and tethers to allow further control of movement through a nanopore, including cis helicase and conjugate moved from cis to trans (pg. 64, ll. 1-2; pg. 82, ll. 12-13; pg. 92, ll. 6-8; pg. 94, l. 17 – pg. 95, l. 3; pg. 66, ll. 5-6; pg. 66, ll. 19-29).
As to the polynucleotide-handling protein is a helicase, a polymerase, an exonuclease, or a topoisomerase in BAYLEY, such proteins were known substitutes for unfoldase, as admitted by Applicants.
Nanopore sensing and characterisation of polypeptides has been proposed in the art. For example, WO 2013/123379 discloses the use of an NTP-driven protein processing unfoldase enzyme to process a protein to be translocated through a nanopore. However, there remains a need for alternative and/or improved methods of characterising polypeptides
(para. 0010).
In addition, as to “leader” terminology, BAYLEY describes the same process as BAI for the polynucleotide therein (pgs. 2-3 & 10-11, describing oligo(dC)30 and charged leader oligonucleotide that drives/threads it into pore). Thus, the charged leader of BAYLEY was a known substitute for the leader of BAI.
In sum, the claims are directed to the combination of familiar nanopore control elements to achieve familiar results, rendering the claims obvious.
Double Patenting- Obvious Type
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 obviousness-type 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); and 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 a nonstatutory double patenting ground provided the conflicting application or patent either is shown to be commonly owned with this application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement.
Effective January 1, 1994, a registered attorney or agent of record may sign a terminal disclaimer. A terminal disclaimer signed by the assignee must fully comply with 37 CFR 3.73(b).
Instant claims 1, 6-11, 13-15, 25, 34, 36 and 38-39 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1, 9-11, 15, 18-20, 23 and 32-38 of 18/357504.
The instant claims are obvious over the conflicting claims because the conflicting claims teach a species of the instant claims. More specifically, the conflicting claims teach:
1. A method, comprising
(i) conjugating a target polypeptide to a polynucleotide to form a polynucleotide- polypeptide conjugate;
(ii) contacting the conjugate with a polynucleotide-handling protein;
(ii) controlling movement of the polynucleotide with respect to a transmembrane protein pore using the polynucleotide-handling protein; and
(iv) taking one or more measurements characteristic of the polypeptide as the conjugate moves with respect to the transmembrane protein pore;
wherein the one or more measurements are indicative of one or more characteristics of the polypeptide selected from: (a) the length of the polypeptide, (b) the identity of the polypeptide, and (c) whether or not the polypeptide comprises post-translational modification,
wherein the conjugate comprises a leader that facilitates threading of the conjugate through the transmembrane protein pore, and wherein the transmembrane protein pore comprises a cis side and a trans side.
It is clear from the face of conflicting claim 1, and dependent claims, that the conflicting claims anticipate the instant claims.
Response to Arguments
Applicants request abeyance, which cannot be granted for substantive rejections. See MPEP § 714.02 (“objections or requirements as to form not necessary to further consideration of the claims” may be held in abeyance).
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Aaron Priest whose telephone number is (571)270-1095. The examiner can normally be reached 8am-6pm.
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/AARON A PRIEST/Primary Examiner, Art Unit 1681
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