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 .
Election/Restrictions
Claims 1-11, 16-19, 21, and 25 are pending and herein discussed on their merits.
Priority
It is acknowledged that the instant application is a 371 of international PCT Application No. PCT/GB/2022/051352, filed May 27, 2022, and that it claims benefit of provisional 63/193,649, filed May 27, 2021, and also claims foreign priority to GB2108936.2, filed June 22, 2021. The effective filing date is considered to be May 27, 2021.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-11, 16-19, 21, and 25 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are drawn to methods of causing enzymatic digestion of nucleic acids using (reversibly inactivated) nucleases. Claim 1 recites the steps of:
combining reagents, in the presence of a temporarily substantially inactive nuclease, to form in vitro synthesized nucleic acid;
detecting, directly or indirectly, the in vitro synthesized nucleic acid; and
subsequent to the detection, permitting or causing the substantially inactive nuclease to regain substantial nuclease activity, such that the in vitro synthesized nucleic acid is digested by the nuclease.
The claimed nuclease is required to possess certain characteristics in order to perform the method as recited. It must be capable of being rendered substantially inactive and then be able to revert to an active state. It must not interfere substantially with the activity of a polymerase, as it is present in a synthesis reaction, and it must be able to digest the synthesized nucleic acid.
In the instant case, the claims do not set forth the nucleases in terms of sufficient relevant identifying characteristics. The claims encompass the use of nucleases which have only been identified in terms of their function. Only claims 4 and 19 describe specific endonucleases (HL-SAN, HL-dsDNase, Cyanase, Benzonase, Cryonase, and OmniCleave).
The size of the genus is very large, encompassing enzymes with wide variety in structure, function, and mechanism, including enzymes which cause single or double-stranded cleavage, which act on DNA or RNA or both, which have different temperature restrictions, and which result in different oligonucleotide products (Yang W. Nucleases: diversity of structure, function and mechanism. Quarterly Reviews of Biophysics. 2011;44(1):1-93.). Claims which restrict the types of nuclease still encompass a large genus – there are many types of nucleases which act on DNA and which require magnesium or manganese ions for activity, for example.
The specification allows for any type of nuclease (par. 21), but examples are provided only for a subset. While the majority of examples in the instant application feature HL-SAN only, Example 2 (par. 53-68; results in Fig. 2) describes characterization of HL-SAN, HL-dsDNase, Cyanase, Benzonase, Cryonase, and OmniCleave relative to their ability to be reversibly inactivated. All nucleases were inactivated using the same protocol (par. 54): 1mM TCEP and incubation at 60ºC for 30min. After this incubation, the enzymes were equilibrated to room temperature for 40min, and the inactivated enzyme was stored at 40ºC for an hour prior to testing (par. 46). Magnesium was then added or not to the inactivated nuclease mixture at 25ºC and activity was assessed after 10min, 24hr, and 48hr (par. 47-48; Figure 2).
The specification provides written description for the following nucleases that are reversibly inactive and can be used in the claimed methods: HL-SAN, HL-dsDNase, Cyanase, Benzonase, Cryonase, and OmniCleave (par. 21). The specification does not describe any other species within the claimed genus to show possession of those species.
Regarding the genus of nucleases, the specification does not describe any structural features of the disclosed reversibly in-activatable nucleases which would have been expected to be shared by members of the claimed genus. The specification does not describe physical and/or chemical characteristics of the disclosed nucleases that would be shared by members of the claimed genus. All members of the genus have the same function, i.e., they are inactive, do not interfere with nucleic acid synthesis, and can be induced to become active and digest nucleic acids, but no correlation between their structure and the common function is disclosed. The level of knowledge and skill in the art does not allow those skilled in the art to structurally envisage or recognize additional members of the claimed genus. Because the structure of the species within the claimed genus is expected to vary unpredictably from the structure of the reversibly in-activatable nucleases disclosed in the specification, the disclosed nucleases are not a “representative number” of species within the claimed genus.
Because the nucleases are not representative of the entire claimed genus, and the specification does not disclose structural features shared by members of the genus, the description of the nucleases would not have put the applicant in possession of common structural attributes or features shared by members of the genus that structurally distinguish the members of the genus from non-members of the genus at the time of filing. Thus, the description of nucleases is not sufficient to describe the claimed genus of reversibly in-activatable nucleases. Accordingly, the specification does not provide a representative number of species or sufficient common structural features to show that the applicant would have been in possession at the claimed genus as a whole at the time of filing.
Regarding the genus of nucleases, the level of knowledge and skill in the art does not allow those skilled in the art to structurally envisage or recognize reversibly in-activatable nucleases, because it is known that enzymes which catalyze DNA and/or RNA cleavage tend to differ unpredictably in terms of their biochemistry (including required cofactors, reaction conditions, substrate and product, and catalytic mechanism). Therefore, those skilled in the art would have recognized that the specification’s disclosure of nucleases: HL-SAN, HL-dsDNase, Cyanase, Benzonase, Cryonase, and OmniCleave would not have put the applicant in possession of any type of nuclease at the time of filing. Thus, the specification does not describe sufficiently detailed, relevant characteristics to show that the applicant was in possession of the claimed genus of any type of nuclease capable of being rendered substantially inactive and able to revert to an active state, which does not interfere substantially with the activity of a polymerase, and is capable of digesting a synthesized nucleic acid.
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-11, 16-19, 21, and 25 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1-11, 16-19, 21, and 25 are rejected as indefinite over the recitation of the term “temporarily” in claims 1, 7, 9, and 16. Although the specification provides examples of “temporarily” inactivating the nuclease in paragraph 19, it not clear whether the definition of the term is limited to those examples. For example, it is not clear whether inactivating a nuclease which subsequently regains activity before an hour or after 48 hours would be considered temporarily inactivated. Therefore, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter so as to avoid infringement.
Claims 1-11, 16-19, 21, and 25 are rejected as indefinite over the recitation of the term “substantially”/”substantial” in claims 1, 7, 10, 16, and 25. Although the specification provides examples of “substantially inactive” nucleases in paragraph 11 and “substantially inactive” nucleases in paragraph 12, the claims are considered indefinite because it is not clear whether “substantially”/”substantial” is limited to those examples. Therefore, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter so as to avoid infringement.
In addition, the recitation of “substantial nuclease activity” in claims 1, 10, and 25 and “digest”/”digestion” in claim 1 is considered unclear. The claims require digestion of the nucleic acid (the activity of the nuclease), and the specification gives describes the nucleic acid being either partially or completely digested (par. 18, 20), but it is not clear what digestion/substantial nuclease activity entails. For example, is degradation by a nicking endonuclease (single-stranded cleavage) considered digestion? Furthermore, the specification provides no clear definition of complete or partial digestion. The specification allows that nucleic acids may be considered completely digested by the nuclease if no fragments longer than 15 nucleotides are detectable by electrophoretic analysis (par. 20), but does not provide any examples of partial digestion. It is therefore unclear how substantial nuclease activity/digestion would be assessed, and one of skill in the art would not be able to determine the metes and bounds of the claimed invention so as to avoid infringement.
Claim 6 is rejected as indefinite over the recitation of the phrase “non-thermal cycling DNA amplification reaction.” This phrase in considered indefinite because “non-thermal cycling DNA amplification reaction” is not clearly defined in the specification and there is no single art recognized definition for this phrase. The specification provides examples of alternative DNA amplification methods which are neither PCR nor isothermal methods in STAR and qSTAR (par. 7, 16). However, it is unclear how such methods would be “non-thermal cycling,” given that they require energy input through heat (and do not instead use an electric field, for example) and cannot be classified as “non-thermal” methods. Furthermore, in the case of qSTAR there is a regularly repeated sequence of temperatures which the reaction moves, and therefore the given embodiments contradict a potential interpretation of the methods as “methods which do not require thermal cycling.” As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter so as to avoid infringement.
Claim 16 is rejected as indefinite over the recitation of “the temporarily substantially reversibly inactivated nuclease.” This phrase lacks antecedent basis because the claim on which it depends (claim 1) does not recite a temporarily reversibly inactivated nuclease, only a “temporarily substantially inactive nuclease.” It is also unclear what is meant by “reversibly,” with no clear definition given in the specification. For example, does a nuclease regaining 84% or less of its activity after inactivation represent a reversibly inactivated nuclease (par. 12)? As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter so as to avoid infringement.
Claim 21 is rejected as indefinite over the recitation of “the solid support.” This phrase lacks antecedent basis because the claims on which it depends do not recite a solid support. It is therefore unclear whether any solid support would meet this limitation, or if there is a particular solid support which is required.
Claim 25 is rejected as indefinite over the recitation of the term “sufficient.” The term is considered indefinite because an amount of magnesium or manganese sufficient to cause the nuclease to regain activity is not clearly defined in the specification. Examples of concentrations of Mg2+ and Mn2+ which lead to inactivity are provided (par. 29), but it is not clear whether “sufficient” amounts for regaining activity are limited to concentrations above those examples. Therefore, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter so as to avoid infringement.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-11, 16-19, 21, and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Lanes et al. (published December 18, 2014; Patent Publication No. 2014/0370514), as evidenced by Columbia University (published November 11, 2020; Columbia University: Recommendations for reducing nucleic acid contamination...; accessed https://research.columbia.edu/sites/research.columbia.edu/files/content/EHS/COVID-19/RecommendationsForReducingNucleicAcidContamination.pdf).
Lanes teaches endonucleases which are useful for removing contaminating nucleic acids from a sample (Abstract), including HL-SAN. Lanes is the Pre-Grant Publication of application 14/377,013 or issued patent 9,422,595, and is mentioned in the instant application’s specification in par. 26.
Regarding claims 1 and 16-17, Lanes teaches combining reagents including DNA polymerase, a plurality of NTPs comprising at least one of dATP, dCTP, dGTP, and dTTP, and a substantially inactive nuclease to synthesize nucleic acid in vitro (par. 29, 136) and detecting the synthesized nucleic acid (par. 6, 136-138).
Lanes does not explicitly teach that the nuclease is reactivated after synthesis to digest the synthesized nucleic acid. However, Lanes does teach activity in nucleases – which have been completely inactivated – following incubation on ice and contact with magnesium ions (par. 128). Although the specification of Lanes describes enzymes being irreversibly inactivated (par. 55-56), they define irreversible in a manner inconsistent with the plain meaning of the term. That is, they define irreversible to include situations in which the continued presence of inactivating conditions is required for lack of activity (par. 55). Lanes discusses factors which affect the ability of nucleases to remain inactive or regain activity in the absence of inactivators (par. 57-58). HL-SAN itself is only taught to be inactivated (even in the absence of inactivation agent) under the following conditions: 10nM TCEP with a 60min incubation at 50ºC followed by removal of TCEP only after storage at room temperature for 2 days (par. 59); 1mM TCEP with a 60min incubation at 50ºC followed by removal of TCEP only after storage at 37ºC for 2 days (par. 60); or 10mM TCEP at 37ºC for 1 day or room temperature for 4 days (par. 61).
Furthermore, data from Lane support an interpretation of the inactivation of the disclosed nucleases which is more in line with the claims of the instant application (that is, reversible for nucleases at the temperature and concentration ranges of the chemicals disclosed). In Example 5, nucleases are shown to be completely inactivated or lacking activity after treatment using 1mM DTT at 40ºC for >5min (Fig. 6a). In Example 7, the same types of enzymes, treated in the same reaction conditions, display substantial activity (Fig. 8b, particularly at lower concentrations and shorter incubations) after being placed on ice and then assayed in the presence of magnesium ions for nuclease activity (par. 128-129). These results are considered to indicate that the nucleases of Lanes are reversibly in-activatable.
Lanes does not teach digestion after synthesis.
The courts have haled that rearrangement of steps is obvious in the absence of unexpected results. Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious).
While Lanes suggests the digestion of nucleic acid prior to synthesis, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to try including this step after synthesis of the nucleic acid. The artisan would have a reasonable expectation of success as the artisan is rearranging known steps. One would be motivated to do so in order to reduce the risk of environmental nucleic acid contamination (Columbia: pg. 3, last paragraph).
Lanes teaches enzymatic digestion in a test device (par. 128). In this case, the Eppendorf tubes are considered to be a general ‘test device.’
Regarding claims 2-4 and 18-19, Lanes teaches the use of endonucleases which act on DNA substrates (par. 2) and require the presence of magnesium or manganese ions (par. 35). Lanes explicitly discusses the use of HL-SAN (Figure 16; par. 104), Benzonase (Figure 16; par. 3, 104), and OmniCleave (par. 3).
Regarding claims 5-6, Lanes discloses the use of DNA amplification reactions, including isothermal techniques (par. 6, 46)
Regarding claims 7-9, Lanes teaches substantial inactivation with reducing agents including DTT and TCEP (par. 56), wherein the nuclease is heat-labile and inactivation is accomplished through incubation between 25-60 ºC for at least 15 minutes (par. 39). Lanes teaches that inactivation with temperature and/or DTT or TCEP can be reversible (par. 127; Figures 8a-d). Figures 8a-d show that significant activity in nucleases is retained after treatment with reducing agents DTT and TCEP and/or temperature inactivation followed by storage on ice (par. 124, 128). In particular, while Figure 6a/b shows almost complete loss of activity in nucleases treated with 1 mM DTT at 40 or 50 ºC for 5 minutes or more, Figure 8a/b show that nucleases treated under the same conditions may retain their ability to cleave target nucleic acids (par. 127).
Regarding claims 10-11 and 25, Lanes teaches contacting the nuclease with magnesium or manganese ions at between 10-100 mM concentrations for activity (par. 35).
Regarding claim 21, Lanes teaches the reagents provided as a liquid (par. 29).
Conclusion
No claims are allowed.
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/C.M.J./Examiner, Art Unit 1682
/AMANDA HANEY/Primary Examiner, Art Unit 1682