METHODS AND COMPOSITIONS FOR PROTEASE REPORTER ASSAYS AND MODULATORS

§103 §112

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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 04/13/2026 has been entered. Priority This Application claims benefit of priority to Provisional Application 63/061,156 filed on 08/04/2020 and Provisional Application 63/209,138 filed on 06/10/2021. This Application is also a 371 of PCT/US2021/043266 filed on 07/27/2021. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Information Disclosure Statement The Information Disclosure Statement filed 04/13/2026 has been acknowledged and considered. Drawings The drawings are objected to because the title of Figure 3 is misspelled. The title reads “Luyciferase Assay” but should read “Luciferase Assay.” Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance. Amendment and Claim Status In the reply filed 04/13/2026, Applicant amended claims 151, 168 and 171. Claims 1-150, 152-156 and 166-167 were previously canceled. Claims 160-165 were previously withdrawn as not being encompassed by the elected group. Claims 151, 157-165 and 168-171 are currently pending. Claims 160-165 remain withdrawn. Claims 151, 157-159 and 168-171 are under examination. Claim Objections Claim 151 is objected to because of the following informalities: Claim 151 recites “an OMA1 reporter comprising” in the preamble of the claim. The first recitation of OMA1 should be preceded by the full name of the protein which OMA1 is shorthand for. Appropriate correction is required. Claims 157-159 are objected to because of the following informalities: Claims 157-159 indicate they are amended, however, the claims do not have any mark-ups and the claims are consistent with the claims filed 11/03/2025. It appears this was inadvertent. Even such, claims 157-159 should read “Previously Presented.” Appropriate correction is required. 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 151, 157-159 and 168-171 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. Claim 151 recites “a domain of at least 75% sequence identity with one sequence selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 27” in part b of the claim. Claim 168 recites “a domain of at least 75% sequence identity with the amino acid sequence: F-W-P-A-R-L-A-T-R-L-L-K-L-R-Y-L-I-L-G-S-A-V-G-G-G-Y-T-A-K-K-T-F (SEQ ID NO: 25)” in part b of the claim. It is noted SEQ ID NO: 25 is the species elected for examination. Claims 157-159 and 169-171 require the OMA1 reporter of either claim 151 or claim 168 and are therefore included in this rejection. The claims are broadly directed to an OMA1 reporter, comprising any OMA-1 cleavable complementation reporter which is being interpreted as any type of reporter, i.e., fluorescent reporter or bioluminescent reporter, both of which are well known in the art, and an amino acid sequence with at least 75% sequence identity to instant SEQ ID NO: 25 or SEQ: 27 wherein an amino acid sequence with at least 75% sequence identity to instant SEQ ID NO: 21 is not included. It is noted an amino acid sequence can have 8 modifications, including conservative and non-conservative substitutions, deletions and insertions and still read on a sequence with at least 75% sequence identity to instant SEQ ID NO: 25. The preamble is interpreted as limiting the reporter to being a functional OMA1 protein or protein fragment, meaning, the OMA1 must still be able to cleave OPA1 as that is the function of OMA1. The written description requirement may be met in light of the state of the art and/or by providing a representative number of species of the genus. With regard to the state of the art, it is well known that even a single amino acid or nucleic acid substitution, or what appears to be an inconsequential chemical modification, will often dramatically affect the biological activity and characteristics of a polynucleotide or polypeptide. Regarding OMA1 specifically, one of the specific sites where OMA1 cleaves OPA1 is between the 194 Arginine (R) and 195 (A) residues (MacMillan-Crow, US 20190049430 A1, Paragraph [0073], Reference of Record). The specific R and A amino acids are required at positions 194 and 195, respectively, for the protein to have activity (MacMillan-Crow, Paragraph [0092]). Also, the sequences immediately flanking the R and A residues are important and activity is decreased when the flanking sequences are changed (MacMillan-Crow, Paragraph [0092]). As such, the art recognizes even a single amino acid change within a sequence of OPA1 can inhibit OMA1 activity. Therefore, the art provides the specific amino acids required for cleaving at that specific site, however, the art does not provide the required amino acids that must be maintained in the claimed sequences in order to maintain the function of being an OMA1 reporter. Thus, the claims are directed to amino acid sequences that can function as OMA1 reporters with no correlated structure associated with that function. Without such structure, the Specification does not convey possession of the breadth of the claimed genus. Alternatively, the written description requirement may be met by providing a representative number of species of the genus. The originally filed claim set, the claim set of the PCT, includes claim 8 which states the targeting sequence has at least 75% sequence identity with any one of a group of sequences which includes SEQ ID NO: 25. However, the only example the Specification provides of a reporter being able to import into the mitochondria is reporter #15 which comprises SEQ ID NO: 15 (Specification, Paragraph [429]). The entirety of SEQ ID NO: 25 is encompassed within SEQ ID NO: 15. Thus, the Specification does not disclose the specific amino acids required to retain functionality because only a reporter with the entire sequence of SEQ ID NO: 25 is disclosed with the function of being able to important into the mitochondria. However, as discussed above, the scope of the claims encompasses sequences with significantly more variability. As discussed above, a sequence can have up to 8 amino acid changes and still read on a sequence with at least 75% sequence identity to instant SEQ ID NO: 25. That is, up to 8 modifications, including conservative and non-conservative substitutions, deletions and insertions, can be made and still read on the instant claims. Thus, the Specification is not sufficient for one of ordinary skill in the art to envisage what modifications to instant SEQ ID NO: 25 can be made with the expectation that the function will be preserved. The Court has interpreted 35 U.S.C. §112, first paragraph, to require the patent specification to “describe the claimed invention so that one skilled in the art can recognize what is claimed” (Enzo Biochem, Inc. v. Gen-Probe Inc, 63 USPQ2d 1609 and 1618) (Fed. Cir. 2002)). In evaluating whether a patentee has fulfilled this requirement, our standard is that the patent’s “disclosure must allow one skilled in the art ‘to visualize or recognize the identity of’ the subject matter purportedly described” (Regents of Univ. of Cal. v. Eli Lilly & Co., 43 USPQ2d 1398 (Fed Cir. 1997)). Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that "Applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the 'written description' inquiry, whatever is now claimed” (See page 1117). In this case, the instant Specification does not "clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed" (See Vas-Cath at page 1116). One cannot describe what one has not conceived. See Fiddes v. Baird, 30 USPQ2d 1481, 1483. Therefore, claims 151, 157-159 and 168-171 do not meet the written description requirement. 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. The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows: 1. Determining the scope and contents of the prior art. 2. Ascertaining the differences between the prior art and the claims at issue. 3. Resolving the level of ordinary skill in the pertinent art. 4. Considering objective evidence present in the application indicating obviousness or nonobviousness. Claims 151, 157-159 and 168-171 are rejected under 35 U.S.C. 103 as being unpatentable over MacMillan-Crow (US 20190049430 A1, 02/14/2019) (Of Record) in view of Reichert et al. (US 20100209436 A1, 08/19/2010). Regarding claims 151 and 168, MacMillan-Crow disclose methods and compositions for detecting mitochondrial dysfunction, specifically through reporter molecules that are cleavable by the zinc metalloprotease metalloendopeptidase OMA1 (OMA1) (See entire document, Abstract). A key regulator in mitochondrial fusion is the optic atrophy protein OPA1 (Paragraph [0071]). Studies show that loss of the long form (L-OPA1) leads to impaired fusion (Paragraph [0071]). MacMillan-Crow further disclose a reporter molecule comprising at least one cleavage site recognized by OMA1, a detection domain capable of emitting a signal and a repressor domain linked to the reporter molecule (Paragraph [0007]). Further, the reporter molecule may comprise functional domains that increase the availability or targeting of the reporter molecule to a cell, cellular organelle or sub-cellular domain, wherein the targeting domain may target the reporter molecule to the mitochondria via a mitochondrial targeting signal (Paragraph [0033]). The reporter comprises a detection domain wherein the detection domain is a fluorescent molecule, wherein the fluorescent molecule is a fluorescent peptide such as a green fluorescent protein (Paragraph [0023]). In one embodiment, MacMillan-Crow disclose the OMA1-dependent processing site is reported to be between the 194 Arginine (R) and the 195 Alanine (A) residues. To test the theory, MacMillan-Crow synthesized a peptide based on the OPA1 sequence that did not include the 194-R and 195-A residues, which showed the 194-R is essential for OMA1 activity (Paragraph [0077]). MacMillan-Crow further disclose additional studies are underway to determine the minimal binding site required for the detection of OMA1 activity (Paragraph [0077]). MacMillan-Crow does not disclose a sequence having at least 75% identity to instant SEQ ID NO. 25. However, Reichert et al. disclose means and methods for therapeutic intervention of mitochondrial disorders or diseases, in particular, a disorder or disease characterized by an altered OPA1 processing (See entire document, Abstract). Mitochondrial dysfunction as observed in a number of human diseases leads to impairment of bioenergetic competence of mitochondria (Paragraph [0220]). The reduction of the membrane potential and/or ATP level in mitochondria leads to a proteolytic breakdown of large isoforms of OPA1 to short isoforms and as a consequence fusion of mitochondria is blocked (Paragraph [0220]). Recovery of mitochondrial fusion was accompanied by resynthesis of large isoforms of OPA1 (Paragraph [0126]). Reichert et al. further disclose a compound capable to modulate fusion or fission of mitochondria within the mitochondrial network, such as an OPA1 isoform (Paragraph [0019]). Based on in silico predictions (prediction of cleavage of the leader sequence by mitochondrial processing peptidase (MPP) using the computer program Mitoprot, it was deduced in a first estimation that large OPA1 isoforms start with amino acid position 95 of all OPA1 spliceforms, and hence, start with the peptide TRLLKLRYLILGS (SEQ ID NO: 25) (Paragraph [0300]). However, based on an alternative MPP cleavage site present from amino acid 86 to 88, it was deduced that large OPA1 isoforms start with amino acid position 88 of all OPA1 spliceforms, and hence, start with the peptide FWPARLATRLLKLRYLILGS (SEQ ID NO: 35) (Paragraph [0300]). Reichert et al. disclose specific large OPA1 isoforms which comprise amino acid stretches or amino acid peptides comprising one or more of the following sequence, two of the sequences being YLILGSAVGGGYTAK (SEQ ID NO: 17) and FWPARLATRLLKLRYLILGS (SEQ ID NO: 25) (Paragraph [0165]). It is noted the combination of SEQ ID NOs: 25 and 17 of Reichert et al. shares 90.3% total sequence identity and 100% local similarity to instant SEQ ID NO: 25. An alignment is provided below wherein Qy is instant SEQ ID NO: 25 and Db is the combination of SEQ ID NOs: 25 and 17 of Reichert et al. It is noted the last six amino acids of SEQ ID NO: 25 of Reichert et al., being YLILGS, overlap with the first six amino acids of SEQ ID NO: 17 Reichert et al. The sequence in the alignment below spans the entire length of SEQ ID NO: 25 of Reichert et al. followed by AVGGGYTAK which is the remaining portion of SEQ ID NO: 17 of Reichert et al. after the overlapping six amino acids (YLILGS) that SEQ ID NO: 25 of Reichert et al. and SEQ ID NO: 17 of Reichert et al. share. PNG media_image1.png 166 640 media_image1.png Greyscale Thus, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized the combination of SEQ ID NOs: 25 and 17 of Reichert et al. in the reporter of MacMillan-Crow as the sequences were known OPA1 isoforms as disclosed by Reichert et al. One of ordinary skill in the art would utilize the combined sequence of Reichert et al. in the reporter of MacMillan-Crow motivated by the desire to promote mitochondrial fusion. As discussed above, Reichert et al. disclose the breakdown of large OPA1 isoforms into short isoforms block mitochondrial fusion, however, mitochondrial fusion can be achieved by resynthesizing the large isoforms. Through computer prediction, Reichert et al. found an alternative MPP cleavage site present from amino acid 86 to 88 and deduced that large OPA1 isoforms start with amino acid position 88 of all OPA1 spliceforms. Thus, it would have been obvious to utilize the combined sequence of the large OPA1 isoforms in the reporter of MacMillan-Crow as it was known large OPA1 isoforms can restore mitochondrial fusion as taught by Reichert et al. It would have been further obvious to utilize the specific combination of SEQ ID NOs: 25 and 17 of Reichert et al. because Reichert et al. found an alternative cleavage site, reading on a binding site, and found that large OPA1 isoforms start with amino acid position 88 of OPA1. Therefore, it would have been obvious to utilize an OPA1 spliceform starting at position 88, meaning the reporter would not comprise the targeting domain of instant SEQ ID NO: 21 as instant SEQ ID NO: 21 comprises the first 66 amino acids of OPA1. Thus, the combination of MacMillan-Crow renders obvious an OMA1 reporter comprising an OMA1-cleavable complementation reporter and a sequence with at least 75% sequence identity to instant SEQ ID NO: 25 wherein the reporter does not comprise a sequence with 75% sequence identity to instant SEQ ID NO: 21. Regarding claims 157 and 169, MacMillan-Crow discloses nucleic acids encoding the reporter molecules disclosed, wherein the nucleic acid can be DNA or RNA and the DNA can be present in a vector (Paragraph [0035]). Further, the nucleic acid sequences may be inserted into a recombinant expression vector (Paragraph [0037]). Regarding claims 158 and 170, MacMillan-Crow discloses a host cell comprising the nucleic acid (Claim 12 of MacMillan Crow) encoding the reporter molecule (Claim 11). Regarding claim 159 and 171, MacMillan-Crow does not explicitly disclose a kit. However, a kit can simply comprise the reporter. Additionally, it is noted the instant Specification states recombinant genes, recombinant protein and recombinant cells or recombinant organisms can be supplied individually, combined or as a kit (Paragraph [278]). Thus, the disclosure of MacMillan-Crow of the reporter, which can be in a recombinant cell, reads on a kit. Response to Arguments Applicant's arguments filed 04/13/2026 have been fully considered but they are not persuasive. It is the Examiner’s position that instant claims 151, 157-159 and 168-171 remain obvious as explained in the modified rejection set forth above. Regarding Applicant’s arguments of unexpected results, it remains the Examiner’s position that the claims are not commensurate in scope with the showing. Figure 3 shows a comparison of Reporter #1 and Reporter #15, with Reporter #15 showing about 67 RLU and Reporter #1 only showing about 1.7 RLU. While this is a significant difference, Reporter #15 is a specific sequence, specifically SEQ ID NO: 27 and a specific sequence encoding an “OMA1-cleavable complementation reporter” as shown on Page 22 of Applicant’s arguments, yet the claims are directed to any potential “OMA1-cleavable complementation reporter,” any sequence that shares 75% sequence identity to instant SEQ ID NO: 25 or 27 and as the claim utilizes open language, meaning “comprising,” the reporter can include any other sequences as long as the sequence doesn’t share at least 75% sequence identity to instant SEQ ID NO: 21. Thus, the claims are much broader in scope than the showing. Conclusion Claims 151, 157-159 and 168-171 are rejected. No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to ASHLEY T WHITE whose telephone number is (571)272-0683. The examiner can normally be reached Monday - Friday 8:30 - 5:00 EST. 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Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /A.T.W./Examiner, Art Unit 1653 /SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653