DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-3 and 6-19 are pending in the application.
Restriction/Election
Applicant's election with traverse of Group I, claims 1-3, 6-14, 18, and 19, in the reply filed August 14, 2026 is acknowledged.
The traversal is on the ground that the da Silva reference is not available as prior art and the shared same or corresponding technical feature is a special technical feature because it makes a contribution over the prior art.
Applicant’s arguments are not found persuasive. The shared same or corresponding technical feature between Groups I and II is a Cre recombinase pro-enzyme, for identifying and quantifying proteolytic activity in a disease or an infection, wherein said cre recombinase pro-enzyme comprising intein mediated circularization, a flip-excision cassette comprising an output effector and/or reporter protein, and a linker sequence cleavable by a protease; wherein the intein mediated circularization is a N-terminal-C-terminal fused split intein mediated circularization; and wherein the cleavable linker sequence comprises from 4-15 amino acids. For reasons set forth in the following rejections under 35 U.S.C. 102 and 35 U.S.C. 103, the shared same or corresponding technical feature between Groups I and II is not a special technical feature as it does not make a contribution over the prior art.
Applicant's election with traverse of flip-excision cassette comprises an output effector protein (species election A), SEQ ID NO: 1, ENLYFQ↓S (species election B), Nostoc punctiforme DnaE split (species election C), virus protease (species election D), and adenovirus virus infection (species election E) in the reply filed August 14, 2026 is acknowledged.
The traversal is on the ground that the da Silva reference is not available as prior art and the shared same or corresponding technical feature is a special technical feature because it makes a contribution over the prior art.
Applicant’s arguments are not found persuasive. For reasons set forth in the following rejections under 35 U.S.C. 102 and 35 U.S.C. 103, the respective shared same or corresponding technical feature among each of the species groupings is not a special technical feature as it does not make a contribution over the prior art.
The requirement is still deemed proper and is therefore made FINAL.
Claims 15-17 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 2 and 10 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 1, 3, 6-9, 11-14, 18, and 19 are being examined on the merits with claims 1, 6, 7, and 11 being examined only to the extent the claims read on the elected subject matter.
Priority
This application is filed under 35 U.S.C. 371 as a national stage of international application PCT/IB202/059356, filed September 21, 2023, which claims foreign priority under 35 U.S.C. 119(a-d) to Portugese application no. 118205, filed September 22, 2022. A certified copy of the foreign priority document has been filed in this application on March 24, 2025.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on June 26, 2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDS has been considered by the examiner.
The listing of references in the specification at pp. 3-4 is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawing Figures
Figure 6 is objected to for disclosing a sequence that is encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2), yet fails to comply with the requirements of 37 CFR 1.821 through 1.825; applicants’ attention is directed to the final rulemaking notice published at 55 FR 18230 (May 1, 1990), and 1114 OG 29 (May 15, 1990). When a sequence is presented in a drawing, regardless of the format or the manner of presentation of that sequence in the drawing, the sequence must still be included in the Sequence Listing and the sequence identifier (“SEQ ID NO:X”) must be used, either in the drawing or in the Brief Description of the Drawings. See MPEP § 2422.02.
To be in compliance, applicants should identify nucleotide sequences of at least 10 nucleotides and amino acid sequences of at least 4 amino acids in the specification by a proper sequence identifier, i.e., “SEQ ID NO:” (see MPEP 2422.01). If these sequences have not been listed in the computer readable form and paper copy of the sequence listing, applicant must provide an initial computer readable form (CRF) copy of the “Sequence Listing”, an initial paper copy of the “Sequence Listing”, as well as an amendment directing its entry into the specification, and a statement that the content of the paper and CRF copies are the same and, where applicable, include no new matter as required by 37 C.F.R. 1.821(e) or 1.821(f) or 1.821(g) or 1.821(b) or 1.825(d).
Specification/Informalities
The substitute specification filed March 24, 2025 is objected to for disclosing sequences that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2), yet failing to comply with the requirements of 37 CFR 1.821 through 1.825; applicants’ attention is directed to the final rulemaking notice published at 55 FR 18230 (May 1, 1990), and 1114 OG 29 (May 15, 1990). See specification at paragraphs [0056], [0073], [0081], [0092], [0094], and Table 1.
To be in compliance, applicants should identify nucleotide sequences of at least 10 nucleotides and amino acid sequences of at least 4 amino acids in the specification by a proper sequence identifier, i.e., “SEQ ID NO:” (see MPEP 2422.01). If these sequences have not been listed in the computer readable form and paper copy of the sequence listing, applicant must provide an initial computer readable form (CRF) copy of the “Sequence Listing”, an initial paper copy of the “Sequence Listing”, as well as an amendment directing its entry into the specification, and a statement that the content of the paper and CRF copies are the same and, where applicable, include no new matter as required by 37 C.F.R. 1.821(e) or 1.821(f) or 1.821(g) or 1.821(b) or 1.825(d).
The substitute specification filed March 24, 2025 is also objected to for disclosing colored features of the drawing figures (e.g. paragraph [0087]), however, the drawings are not in color. Appropriate correction is required.
Claim Objections
Claims 6, 7, 11, and 13 are objected to because of the following informalities:
Claim 6 is objected to in the recitation of “the linker sequence cleavable” and in the interest of improving claim form, it is suggested that the noted phrase be amended to recite “the cleavable linker sequence.”
Claim 6 is also objected to in the recitation of “SEQ. ID. 1” and in the interest of improving claim form, it is suggested that all instances of “SEQ. ID.” be replaced with the preferred sequence identifier “SEQ ID NO:”. See 37 CFR 1.821(d).
Claim 6 is also objected to as failing to recite a conjunction between the alternatives and in the interest of improving claim form, it is suggested that the conjunction “or” be inserted immediately before “SEQ. ID. 17.”
Claim 7 is objected to in the recitation of “the split inteins is” and in the interest of improving claim form, it is suggested that the noted phrase be amended to recite “the split intein is.”
Claim 11 is objected to in the recitation of “a adenovirus virus infection” and in the interest of improving claim form, it is suggested that the noted phrase be amended to recite “an adenovirus virus infection.”
Claim 13 is objected to in the recitation of “proteolytic activity of recombinase” and in the interest of improving claim form, it is suggested that the noted phrase be amended to delete the phrase “of recombinase.”
Claim Rejections - 35 USC § 112(b)
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 1, 3, 6-9, 11-14, 18, and 19 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 1 (claims 3, 6-9, 11-14, 18, and 19 dependent therefrom) is confusing in the recitation of “wherein said cre recombinase pro-enzyme comprising…a flip-excision cassette comprising an output effector.” According to the instant specification, the recited “flip-excision cassette” corresponds to a “FLEx reporter module,” which is a polynucleotide and is separate from the recited “Cre recombinase pro-enzyme” (see paragraphs [0041] and [0067] and Figure 2). It is suggested that applicant clarify the scope of the claimed Cre recombinase pro-enzyme.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 3, 8, 9, 11-14, and 18 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ploegh et al. (US 2011/0321183 A1; cited on the attached Form PTO-892; hereafter “Ploegh”).
Regarding claim 1, Ploegh teaches a circularized version of a polypeptide, wherein the original C- and N-termini of the polypeptide are joined to each other via a linker peptide (paragraph [0028]). Ploegh teaches that a circularized polypeptide displays at least one enhanced property relative to a non-circular form including increased stability, e.g., increased resistance to exoprotease(s), increased thermal stability, increased circulating time in vivo, increased biological half-life, less propensity to aggregate, and/or increased rate of refolding (paragraph [0287]). Specifically, Ploegh teaches N-terminal to C-terminal sortase-mediated circularization of Cre recombinase (paragraphs [0052], [0053], and [0491]). Ploegh teaches the circularized Cre recombinase comprises a sortase-cleavable LPETG motif (paragraph [0491]). The sortase-cleavable LPETG motif is considered to be encompassed by “a linker sequence cleavable by a protease.”
The recitation of “for identifying and quantifying proteolytic activity in a disease or an infection” in claim 1 is interpreted as reciting an intended use or purpose of the claimed Cre recombinase pro-enzyme (see MPEP 2111.02.II regarding preamble statements reciting purpose or intended use). In this case, the recitation of “for identifying and quantifying proteolytic activity in a disease or an infection” in claim 1 does not limit the structure and/or function of the claimed Cre recombinase pro-enzyme.
The recitation of “wherein said cre recombinase pro-enzyme comprising intein mediated circularization…wherein the intein mediated circularization is a N-terminal-C-terminal fused split intein mediated circularization” in claim 1 is interpreted as a “product-by-process” limitation (see MPEP 2113 regarding product-by-process claims). According to MPEP 2113.I, the patentability of a product does not depend on its method of production, if the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process. In this case, even though the circularized cre recombinase of Ploegh is made by a different process, the structure of the circularized cre recombinase is the same as and is encompassed by the Cre recombinase pro- enzyme of claim 1.
Regarding claim 3, as stated above, according to the disclosure of the specification, the claimed Cre recombinase pro-enzyme is separate from and excludes a “flip-excision cassette.” Given a broadest reasonable interpretation in view of the indefiniteness of the phrase ““wherein said cre recombinase pro-enzyme comprising…a flip-excision cassette comprising an output effector” in claim 1 and in light of the disclosure of the specification, claim 3 is included in the instant rejection based on the interpretation of claim 1 as excluding “a flip-excision cassette comprising an output effector and/or reporter protein.”
Regarding claims 8, 9, and 11, the recitation of “wherein the proteolytic activity is a virus activity or cellular activity” in claim 8, “wherein the protease is a virus protease” in claim 9, and “wherein the infection is…a adenovirus virus infection” in claim 11 are in reference to the intended use limitation “for identifying and quantifying proteolytic activity in a disease or an infection” in claim 1. These limitations are interpreted as statements reciting purpose or intended use and do not limit the structure and/or function of the claimed Cre recombinase pro-enzyme.
Regarding claims 12-14, the only required component of the composition, kit, and virus detection sensor of claims 12-14 is the pro-enzyme of claim 1. Given a broadest reasonable interpretation, the reaction mixture comprising a circularized Cre recombinase of Ploegh (paragraphs [0052] and [0053]) is considered to be encompassed by claims 12-14. The recitation of “for use in detecting viruses and/or proteolytic activity of recombinase” in claim 13 is interpreted as a statement reciting purpose or intended use and does not limit the structure and/or function of the recited Cre recombinase pro-enzyme and kit.
Regarding claim 18, Ploegh teaches the circularized Cre comprises a sortase-cleavable LPETG motif (paragraph [0491]). The sortase-cleavable LPETG motif is considered to be encompassed by “a linker sequence cleavable by a protease.”
Therefore, Ploegh anticipates claims 1, 3, 8, 9, 11-14, and 18 as written.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 3, 6, 8, 9, 11-14, 18, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Ploegh in view of Waugh et al. (Protein Expression and Purification 80:28-293, 2011; cited on the attached Form PTO-892; hereafter “Waugh”).
The relevant teachings of Ploegh as applied to claims 1, 3, 8, 9, 11-14, and 18 are set forth above.
Regarding claims 6 and 19, Ploegh further teaches a cleavage site for a protease that is located at a position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids away from an end of the sortase recognition sequence (paragraph [0278]). Ploegh teaches a number of proteases that cleave polypeptides at particular sequences are known in the art (paragraph [0278]).
Ploegh does not teach the cleavage sequence of ENLYFQ↓S in claim 6 and a cleavable linker sequence comprising from 6 to 10 amino acids in claim 19.
Waugh teaches proteases and their corresponding cleavage sites for cleavage of fusion proteins and affinity tags (p. 284, column 1 and Table 1) including TEV protease, which cleaves the sequence ENLYFQ↓S (p. 284, Table 1; p. 286, column 1).
In view of the combined teachings of Ploegh and Waugh, it would have been obvious to one of ordinary skill in the art before the effective filing date for a circularized cre recombinase comprising the TEV protease cleavage site of Waugh. One would have been motivated and would have expected success for a circularized cre recombinase comprising the TEV protease cleavage site of Waugh because Ploegh taught circularizing a polypeptide to impart beneficial properties including circularizing a cre recombinase, Ploegh taught including a protease cleavage site but did not teach or suggest a particular protease cleavage site, and Waugh taught proteases and their corresponding cleavage sites for cleavage of fusion proteins and affinity tags including TEV protease, which cleaves the sequence ENLYFQ↓S.
Therefore, claims 1, 3, 6, 8, 9, 11-14, 18, and 19 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 1, 3, 6-9, 11-14, 18, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Hsieh et al. (PLoS ONE 12:e018870, 2017 and Supplementary Figure 4, 20 pages; cited on the attached Form PTO-892; hereafter “Hsieh”) in view of Ploegh, Guerreiro et al. (ACS Sens. 4:1654-1661, 2019; cited on the IDS filed June 26, 2025; hereafter “Guerreiro”), and Schnütgen et al. (Nature Biotechnology 21:562-565, 2003; cited on the IDS filed June 26, 2025; hereafter “Schnütgen”).
Regarding claims 1, 3, and 7-9, Hsieh teaches a biosensor system for detection and quantification of dengue virus (title). The biosensor system comprises i) a sensor module with a dengue virus cleavage site and a Cre recombinase and ii) a reporter comprising loxP-flanked mCherry and a mHRP cDNA (p. 1, middle) to switch between mCherry expression in the absence of Cre and mHRP expression in the presence of Cre. Given a broadest reasonable interpretation, the recitation of “output effector protein” in claims 1 and 3 encompasses the mHRP of Hsieh. Hsieh teaches that in the presence of dengue virus, the sensor module is cleaved by the dengue viral protease allowing the Cre recombinase to translocate to the nucleus and trigger the reporter system to excise mCherry due to the flanking loxP sites and express mHRP (p. 1, middle; p. 11, bottom; Figure 5).
The differences between Hsieh and claims 1, 3, and 7-9 are:
Hsieh does not teach intein-mediated circularization of the sensor module; and
Hsieh does not teach a flip-excision (FLEx) cassette comprising the mCherry and mHRP genes.
Regarding difference 1), Ploegh teaches a circularized version of a polypeptide, wherein the original C- and N-termini of the polypeptide are joined to each other via a linker peptide (paragraph [0028]). Ploegh teaches that a circularized polypeptide displays at least one enhanced property relative to a non-circular form including increased stability, e.g., increased resistance to exoprotease(s), increased thermal stability, increased circulating time in vivo, increased biological half-life, less propensity to aggregate, and/or increased rate of refolding (paragraph [0287]). Ploegh teaches sortase-mediated circularization of Cre recombinase (paragraphs [0052], [0053], and [0491]).
Guerreiro teaches a cyclized viral sensor comprising an adenoviral protease cleavage site and a green fluorescent protein (p. 1654, Abstract; p. 1655, column 1, middle). Guerreiro teaches the viral sensor was cyclized by means of protein cyclization promoted by the efficient Nostoc punctiforme Dna intein (Npu DnaE) (p. 1655, column 1, middle; p. 1656, paragraph bridging columns 1-2 and Figure 1; p. 1657, column 2).
In view of the combined teachings of Hsieh, Ploegh, and Guerreiro, it would have been obvious to one of ordinary skill in the art before the effective filing date to circularize the viral sensor module of Hsieh by Npu DnaE-mediated circularization. One would have been motivated to circularize the viral sensor module of Hsieh by Npu DnaE-mediated circularization because the viral sensor module of Hsieh is non-circular while Ploegh taught that a circularized polypeptide displays at least one enhanced property relative to a non-circular polypeptide and Guerreiro taught Npu DnaE-mediated circularization for cyclizing a viral sensor polypeptide. One would have had a reasonable expectation of success because the viral sensor module of Hsieh comprises Cre recombinase and is cleaved by a viral protease, Ploegh taught circularization of Cre recombinase, and Guerreiro taught a method for Npu DnaE-mediated circularization of a viral sensor polypeptide, which is cleaved by a viral protease.
Regarding difference 2), Schnütgen teaches a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on (p. 562, Abstract). Schnütgen illustrates the FLEx switch by a plasmid with EGFP in the sense orientation and a promoterless lacZ (β-galactosidase) in the antisense orientation for expression of EGFP in the absence of Cre and expression of lacZ in the presence of Cre (p. 562, columns 1-2 and Figure 1).
In view of the combined teachings of Hsieh and Schnütgen, it would have been obvious to one of ordinary skill in the art before the effective filing date to substitute the genetic switch of the reporter of Hsieh according to the FLEx switch concept of Schnütgen. One would have been motivated and would have expected success to make the substitution because Hsieh taught a Cre-dependent genetic switch through which the expression of a first given gene (mCherry) is turned off, while the expression of another one (mHRP) is concomitantly turned on and Schnütgen taught an alternative Cre-dependent genetic switch (FLEx switch) through which the expression of a first given gene is turned off, while the expression of another one is concomitantly turned on. One of ordinary skill would have found it obvious to make the substitution because, based on the relevant teachings of Hsieh and Schnütgen, an ordinarily skilled artisan would have predicted that a FLEx switch would be equivalent to the genetic switch of Hsieh.
Regarding claim 6, Hsieh teaches detection of Zika virus (p. 11, Figure 4), however, the expression of EGFP was lower than that observed with dengue virus (p. 8, bottom). Hsieh acknowledges that the Zika virus protease cleavage site is different from the dengue virus protease cleavage site (p. 13, bottom; Supplementary Figure 4). In view of the teachings of Hsieh, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the TNTRR↓GTGNIG protease cleavage site of the sensor module of Hsieh to LVKRR↓GGGTG to increase detection sensitivity of Zika virus. One would have been motivated and would have expected success to do this because while Hsieh taught Zika virus could be detected using the dengue virus protease cleavage site TNTRR↓GTGNIG, the expression of EGFP was lower than that observed with dengue virus, and Hsieh taught the protease cleavage sites of Zika virus and dengue virus are different with Zika virus protease cleaving the sequence LVKRR↓GGGTG.
Regarding claim 11, Hsieh teaches detection of Zika virus (p. 11, Figure 4).
Regarding claims 12-14, the only required component of the composition, kit, and virus detection sensor of claims 12-14 is the pro-enzyme of claim 1. Hsieh teaches a cell comprising the biosensor system (p. 4 and Figure 1) and given a broadest reasonable interpretation, a cell comprising the biosensor system of Hsieh modified according to Ploegh, Guerreiro, and Schnütgen as described above is considered to be encompassed by claims 12-14.
Regarding claims 18 and 19, Hsieh teaches the sensor module comprises the DENV-2 NS4 and the 10 amino terminal amino acids of NS5 (p. 1, middle), which, as shown by Supplementary Figure 4 of Hsieh, includes the 11 amino acid sequence TNTRR↓GTGNIG cleaved by dengue virus protease.
Therefore, the Cre recombinase pro-enzyme of claims 1, 3, 6-9, 11-14, 18, and 19 would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
In the interest of clarity, it is noted that the instant rejection is directed to the non-elected species LVKRR↓GGG in claim 6 and the non-elected species of Zika virus in claim 11. The non-elected species have yet to be searched and examined on the merits because the cited prior art was identified during a search and examination limited to the elected species.
Citation of Relevant Prior Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Iwai et al. (FEBS Lett. 580:1853-1858, 2006; cited on the attached Form PTO-892) teaches highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme (title).
Conclusion
Status of the claims:
Claims 1-3 and 6-19 are pending.
Claims 2, 10, and 15-17 are withdrawn from consideration.
Claims 1, 3, 6-9, 11-14, 18, and 19 are rejected.
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DAVID J STEADMAN whose telephone number is (571)272-0942. The examiner can normally be reached Monday to Friday, 7:30 AM to 4:00 PM.
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/David Steadman/Primary Examiner, Art Unit 1656
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