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
Applicant’s election without traverse of Group I (claims 1-7, 10-11, 30-36, 45-46, and 116) and of Species Group A: a Src homology-3 (SH3) domain and Species Group B: a peptide capable of binding to SH3 domain in the reply filed on April 15, 2026 is acknowledged.
Claims 94 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group II of invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on April 15, 2026.
Accordingly, claims 1-7, 10-11, 30-36, 45-46, and 116 are examined herein.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 120 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63/031,388, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. The full scope of instant claim 1 is not supported by ‘388. Specifically, instant claim 1 recites a first fusion protein and an additional fusion protein comprising (a) or (b). Although ‘388 discloses each individual component (e.g., first, second, and third DNA construct), ‘388 discloses these components only in the context of a first fusion protein and an additional fusion protein comprising (a) and (b). Accordingly, the scope ‘388 is different than instant claim 1.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Claims 1-7, 10-11, 30-36, 45-46, and 116 are granted priority to the International Patent Application PCT/US2021/034570 filed on 05/27/2021.
Drawings
The drawings are objected to because 37 CFR 1.84 (u)(1) states “Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter.” In the current case, the view numbers for the partial views for Figure 2 that appear on several sheets are followed by "Cont." instead of a capital letter such as FIG. 1A, FIG. 1B, etc.
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.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - The Incorporation by Reference paragraph required by 37 CFR 1.821(c)(1) is missing or incomplete. See item 1) a) or 1) b) above.
The name of the ASCII text file and the date of creation are incorrect, and the size of the ASCII text file is currently not in bytes.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Objections
Claims 2, 5, 31, and 32 are objected to because of the following informalities:
Claim 2 recites “form an assembled phase, the assemble phase comprising at least one aggregate” in line 2. A wherein clause is missing further limit the assembled phase. It is recommended to amend claim to “form an assembled phase, wherein the assembled phase comprises at least one aggregate”.
Claim 5 recites “fusion protein a stimulus selected” in line 2. There is a preposition missing between the words protein and a stimulus. It is recommended to amend claim to recite “upon exposure of said at least one additional fusion protein to a stimulus selected from the group”.
Appropriate correction is required.
Applicant is advised that should claim 31 be found allowable, claim 32 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
In this case, claim 1 recites “wherein the peptide ligand is capable of binding to the at least one PPID” in line 10; therefore, its dependent claims 31 and 32 already comprises this limitation. The scope of claim 31 is directed specific species of PPID of claim 1 by the recitation of “wherein the PPID is selected from the group” in lines 1-2. Although claim 32 recites “wherein the peptide is a peptide capable of binding to a PPID” in lines 1-2, it is reminded that this limitation is already included in the claim which it depends. Therefore, the scope of claim 32 is also directed to specific species of PPID of claim 1, which covers the same scope of claim 31.
Claim Rejections - 35 USC § 112
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 2 and 7 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.
Claim 2 recites “wherein the composition is configured to form an assembled phase”, which renders the determination of the exact scope of the claim difficult. Claim 2 does not further recite any concrete structure or measurable properties. With the recitation of “configured to form”, it is unclear whether the composition inherently forms an assembled phase, merely has the potential to form an assembled phase, forms an assembled phase under specific conditions, or must be in an assembled phase.
The term “increases” in claim 7 is a relative term which renders the claim indefinite. The term “increases” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear which specific degree is considered to be increases production of at least one chemical in the cell.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 4 and 45 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim 4 recites “wherein the peptide ligand binds to the PPID”, which is a limitation already included in claim 1 from which claim 4 depends. Further, claim 4 recites “thereby recruiting the target protein to the phase-separated clusters” which describes a behavior resulting from the binding activity of the peptide ligand and the PPID and does not further limit the structure of neither peptide ligand or PPID. In addition, this recitation describes an inherent property and does not further limit the subject matter of the claim from which it depends and includes all of the limitations of that claim. It is recited in claim 1 that peptide ligand is fused to a target protein in line 2, therefore, the target protein is inherently “recruited” to wherever the peptide ligand and PPID are.
Claim 45 is of improper dependent form because it fails to further limit the subject matter of the claim from which it depends and includes all of the limitations of that claim. Claim 1 is directed to a composition of fusion proteins, but dependent claim 45 is directed to nucleic acids.
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.
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.
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 -7, 10-11, 30-36, 45-46, and 116 are rejected under 35 U.S.C. 103 as being unpatentable over Bracha et al (Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds; Cell, 2018, 175:1467-1480; IDS received on 11/28/2022, Non-Patent Literature Cite No. 2) in view of Dueber et al (Synthetic protein scaffolds provide modular control over metabolic flux; Nat Biotech, 2009, 27 (8):753-759), Lin et al (Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs; J. Biol. Chem., 2017, 292(46):19110-19120) and McGee et al (Identification of an Intramolecular Interaction between the SH3 and Guanylate Kinase Domains of PSD-95; Journal of Biological Chemistry, 1999, 274(25:17431-17436).
Regarding claims 1, 10, 11, 30-36, and 116, Bracha et al teach the composition comprises: at least one fusion protein comprising: a second fusion protein comprising a self-assembling protein (e.g., human ferritin heavy chain (FTH1) protein subunits that self-assemble to form a spherical particle) and iLIDs which strongly heterodimerizes with its cognate partner SspB in response to blue light activation; and a third fusion protein comprising an intrinsically disordered protein region (IDR) (e.g., FUS) (pg. 1469, col. 2, para. 2-3). Bracha et al further teaches the second and third fusion proteins each comprises a fluorescent tag or target protein covalently linked, e.g., EGFP and mCherry, (Fig. 2, annotated below). Bracha et al teaches “as many as 24 IDRs are induced to directly assemble on each Core”, which is comprised of another “24 human ferritin heavy chain protein subunits that also self-assemble to form a spherical particle of 12 nm diameter” (pg. 1469, col. 2, para. 2-3). Bracha et al positively teach that Cores must recruit up to 48 partners simultaneously.
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However, Bracha et al teach wherein either the second or third fusion protein comprises at least one PPID and that the target protein is fused to a peptide ligand, wherein the peptide ligand is capable of binding to at least one PPID.
Dueber et al teach fusion proteins comprising a Src homology 3 domain (SH3) and SH3 interaction domains “provided control over the relative abundances of these two enzymes in the resultant complex” because “the relative stoichiometries of these two enzymes in the complex were controlled by varying the number of interaction ligands tethered to [each enzyme] (pg. 755, col. 1, para. 1). Dueber et al further demonstrates that production yield increased as the number of SH3 ligands recombined to each enzyme was increased (Figure 2b and 2c).
In addition, Lin et al teach fusion proteins comprising intrinsically disordered regions (IDRs), specifically FUS protein, and multivalent SH3 domain that bind to a poly-proline-rich-motif ligand (polyPRM) (pg. 19111, col. 1, para. 3). Lin et al further teach that FUS IDRs connected to multivalent SH3 domains retained their ability to undergo liquid-liquid phase separation (Figures 1B and 1C).
In addition, McGee et al teach fusion proteins comprising GFP and protein-protein interaction motifs including PDZ, SH3, and guanylate kinase (GK) (abstract, FIG. 1A). McGee et al further teach GFP is successfully recruited to glutathione S-transferase (GST) because “GFP-GK binds specifically to GST-SH3 [and] GFP-SH3 binds to GST-GK” via interactions between SH3 and GK (FIG. 1B, brief description of the figure).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the rigid covalent linker connecting the target protein or fluorescent tag in the fusion proteins of Bracha et al to comprise a flexible and modular protein-peptide interaction domain taught by Dueber et al. This modification would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted component (SH3 and SH3 interaction domains) and its function (mediating protein scaffold and noncovalently linking two or more proteins) were known in the art. One would have been motivated to have done so for the advantage of avoiding obstruction of IDRs or the Core by the covalently connected fluorescent tags, providing orientation flexibility between IDRs and the Core, improve IDRs flexibility to engage with neighboring IDRs, reducing steric hindrance, and gaining control over the relative abundances of fluorescent tags in the complex as taught by Dueber et al. One would have had a reasonable expectation of success in doing so because Dueber et al, Lin et al, and McGee et al SH3 and its interaction domains offer predictable protein-peptide interactions; Lin et al demonstrates that IDRs, especially FUS proteins, fused to multivalent SH3 function predictably; and McGee et al demonstrates that GFP fused to SH3 or its interaction domain GK also functions predictably.
Regarding claims 2-3, and 5, the specification discloses “an assembled phase means formation of a separate phase…formation of liquid-liquid phase-separated clusters” and “phase-separated cluster refers to an agglomerate colonized by the interaction between particles formed by self-assembly of the self-assembled proteins” ([0094] and [0095]). Bracha et al teach “in response to blue light activation, as many as 24 IDRs are induced to directly assemble on each Core, thus rapidly forming self-interacting particles”, and they condensate as liquids with an “ability to rapidly fuse with one another and round up upon contact” (Figure 1D, 1E, and 1F; pg. 1469, col. 2, para. 3). Bracha et al also refers them as “phase separated liquid condensates” (pg. 1470, col. 2, para. 1).
Regarding claim 4, Bracha et al the fusion proteins comprising IDRs and ferritin rapidly form self-interacting particles that result in phase-separated clusters (see discussion above as applied to claim 3). If the target protein (e.g., GFP) is fused to the peptide ligand (e.g., GK that is a SH3 interaction domain), and one of the fusion proteins (e.g., iLIDs and FTH1) by applying the obviousness to modify the covalent linker in fusion proteins of Bracha et al to SH3 domain as discussed above in claim 1, it is inherent that the target protein will be recruited to the phase-separated clusters upon the peptide ligand binding to the protein-peptide interaction domain (e.g., SH3).
Regarding claim 7, Bracha et al teach little to no phosphorylation are observed on FUS IDR in unstressed cells, but “spatiotemporal changes to the average [post-translational modifications] state, for example under stress, during development or through the cell cycle, provide the cell with a set of handles to dynamically structure these phase diagrams for particular functional requirements” (pg. 1477, col. 2, para. 1). Therefore, Bracha et al positively suggest that the presence of phase separated liquid condensates increases production of at least one chemical in the cell (e.g., phosphorylation).
Regarding claims 6, 45, and 46, Bracha et al teach nucleic acid constructs encoding each fusion protein recited in claim 1 (pg. e4, para. 1 and 2). Further, Bracha et al teach these nucleic acids are introduced into lentiviruses (i.e., engineered system) for following transfection in HEK293T cells (pg. e4, para. 3).
Double Patenting
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 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); 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 nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 3, 6, 10-11, 33-34, and 36 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-5, and 9 of U.S. Patent No. 11,053,492 B2 (hereinafter as ‘492) in view of Dueber et al (Synthetic protein scaffolds provide modular control over metabolic flux; Nat Biotech, 2009, 27 (8):753-759), Lin et al (Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs; J. Biol. Chem., 2017, 292(46):19110-19120) and McGee et al (Identification of an Intramolecular Interaction between the SH3 and Guanylate Kinase Domains of PSD-95; Journal of Biological Chemistry, 1999, 274(25:17431-17436).
Regarding instant claims 1 and 10-11, ‘492 teaches a composition comprising a plurality of fusion proteins, wherein each fusion protein comprises: a first fusion protein comprising a target protein, a second fusion protein comprising a self-assembling protein, and a third fusion protein comprising a low complexity or intrinsically disordered protein region (IDR). ‘492 further teaches wherein the target protein is attached to a C-terminus of the IDR, and the composition further comprises a fluorescent tag (claims 1 and 5).
However, ‘492 does not teach wherein the fluorescent tag is the target protein, and wherein the protein is fused to a peptide ligand that is capable of binding to at lease one protein-peptide interaction domain in the second or third fusion protein.
Dueber et al teach fusion proteins comprising a Src homology 3 domain (SH3) and SH3 interaction domains “provided control over the relative abundances of these two enzymes in the resultant complex” because “the relative stoichiometries of these two enzymes in the complex were controlled by varying the number of interaction ligands tethered to [each enzyme] (pg. 755, col. 1, para. 1). Dueber et al further demonstrates that production yield increased as the number of SH3 ligands recombined to each enzyme was increased (Figure 2b and 2c).
In addition, Lin et al teach fusion proteins comprising intrinsically disordered regions (IDRs), specifically FUS protein, and multivalent SH3 domain that bind to a poly-proline-rich-motif ligand (polyPRM) (pg. 19111, col. 1, para. 3). Lin et al further teach that FUS IDRs connected to multivalent SH3 domains retained their ability to undergo liquid-liquid phase separation (Figures 1B and 1C).
In addition, McGee et al teach fusion proteins comprising GFP and protein-protein interaction motifs including PDZ, SH3, and guanylate kinase (GK) (abstract, FIG. 1A). McGee et al further teach GFP is successfully recruited to glutathione S-transferase (GST) because “GFP-GK binds specifically to GST-SH3 [and] GFP-SH3 binds to GST-GK” via interactions between SH3 and GK (FIG. 1B, brief description of the figure).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the rigid covalent linker connecting the target protein or fluorescent tag in the fusion proteins of ‘492 to comprise a flexible and modular protein-peptide interaction domain taught by Dueber et al. This modification would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted component (SH3 and SH3 interaction domains) and its function (mediating protein scaffold and noncovalently linking two or more proteins) were known in the art. One would have been motivated to have done so for the advantage of avoiding obstruction of IDRs or the Core by the covalently connected fluorescent tags, providing orientation flexibility between IDRs and the Core, improve IDRs flexibility to engage with neighboring IDRs, reducing steric hindrance, and gaining control over the relative abundances of fluorescent tags in the complex as taught by Dueber et al. One would have had a reasonable expectation of success in doing so because Dueber et al, Lin et al, and McGee et al SH3 and its interaction domains offer predictable protein-peptide interactions; Lin et al demonstrates that IDRs, especially FUS proteins, fused to multivalent SH3 function predictably; and McGee et al demonstrates that GFP fused to SH3 or its interaction domain GK also functions predictably.
Regarding instant claim 3, ‘492 further teaches wherein the fusion proteins are configured to form phase separated clusters (claim 1)
Regarding instant claim 6, ‘492 further teaches wherein the composition is present in a cell (claim 9)
Regarding instant claims 33-34, and 36, ‘492 further teaches wherein the self-assembling protein is a ferritin heavy or light chain, and wherein the IDR is selected from the group consisting of FUS or FUSn (claims 2-4)
Claims 1, 2, 6, 10-11, 33-34, and 36 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 8, 10-11, and 13 of U.S. Patent No. 10,538,756 B2 (hereinafter as ‘756) in view of Bracha et al (Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds; Cell, 2018, 175:1467-1480; IDS received on 11/28/2022, Non-Patent Literature Cite No. 2), Dueber et al (Synthetic protein scaffolds provide modular control over metabolic flux; Nat Biotech, 2009, 27 (8):753-759), Lin et al (Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs; J. Biol. Chem., 2017, 292(46):19110-19120) and McGee et al (Identification of an Intramolecular Interaction between the SH3 and Guanylate Kinase Domains of PSD-95; Journal of Biological Chemistry, 1999, 274(25:17431-17436).
Regarding instant claims 1 and 10-11, ‘756 teaches a composition comprising: a second fusion protein comprising a self-assembling protein and a third fusion protein comprising a low complexity or intrinsically-disordered protein region (IDR) (claim 1). ‘756 further teaches wherein the second fusion protein further comprises a fluorescent tag (claim 10).
However, ‘756 does not teach a first fusion protein comprising a target protein that is a fluorescent protein and that it is fused to a peptide ligand capable of binding to at least one PPID.
Bracha et al teach the same composition of ‘756 comprising a second and third fusion proteins (pg. 1469, col. 2, para. 2-3). Bracha et al further teaches the second and third fusion proteins each comprises a fluorescent tag (i.e., target protein) covalently linked to the second and third fusion protein to aid visualization and track formation of phase-separated clusters (Fig. 2).
In addition, Dueber et al teach fusion proteins comprising a Src homology 3 domain (SH3) and SH3 interaction domains “provided control over the relative abundances of these two enzymes in the resultant complex” because “the relative stoichiometries of these two enzymes in the complex were controlled by varying the number of interaction ligands tethered to [each enzyme] (pg. 755, col. 1, para. 1). Dueber et al further demonstrates that production yield increased as the number of SH3 ligands recombined to each enzyme was increased (Figure 2b and 2c).
In addition, Lin et al teach fusion proteins comprising intrinsically disordered regions (IDRs), specifically FUS protein, and multivalent SH3 domain that bind to a poly-proline-rich-motif ligand (polyPRM) (pg. 19111, col. 1, para. 3). Lin et al further teach that FUS IDRs connected to multivalent SH3 domains retained their ability to undergo liquid-liquid phase separation (Figures 1B and 1C).
In addition, McGee et al teach fusion proteins comprising GFP and protein-protein interaction motifs including PDZ, SH3, and guanylate kinase (GK) (abstract, FIG. 1A). McGee et al further teach GFP is successfully recruited to glutathione S-transferase (GST) because “GFP-GK binds specifically to GST-SH3 [and] GFP-SH3 binds to GST-GK” via interactions between SH3 and GK (FIG. 1B, brief description of the figure).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified composition of ‘756 to comprise a fluorescent protein in a first fusion protein as taught by Bracha et al via flexible and modular protein-peptide interaction domains as taught by Dueber et al. This modification would have merely amounted to a simple combination of prior art elements to yield predictable results. Each combined element (fluorescent protein and ‘756 fusion proteins) merely performs the same function as it does separately. The modification of the covalent linker in Bracha et al to SH3 interaction domains would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted component (SH3 and SH3 interaction domains) and its function (mediating protein scaffold and noncovalently linking two or more proteins) were known in the art. One would have been motivated to have done so for the advantage of using fluorescent proteins to visualize spatiotemporal formation of phase-separated clusters from the second and third fusion proteins, using SH3 interaction domains to avoid obstruction of IDRs or the Core by the covalently connected fluorescent tags, and ultimately providing orientation flexibility between IDRs and the Core, improve IDRs flexibility to engage with neighboring IDRs, reducing steric hindrance, and gaining control over the relative abundances of fluorescent tags in the complex as taught by Dueber et al. One would have had a reasonable expectation of success in doing so because Bracha et al already teach a composition comprising the second and third fusion proteins of ‘756 with the presence of fluorescent proteins. One would have also had a reasonable expectation of success in doing so because Dueber et al, Lin et al, and McGee et al SH3 and its interaction domains offer predictable protein-peptide interactions; Lin et al demonstrates that IDRs, especially FUS proteins, fused to multivalent SH3 function predictably; and McGee et al demonstrates that GFP fused to SH3 or its interaction domain GK also functions predictably.
Regarding instant claim 2, ‘756 further teaches wherein the composition forms an assembled phase comprising aggregates (claim 13).
Regarding instant claim 6, ‘756 further teaches wherein the composition is present in a cell (claim 11).
Regarding instant claims 33-34, and 36, ‘756 further teaches wherein the self-assembling protein is a ferritin heavy or light chain, and wherein the IDR is selected from the group consisting of FUS or FUSn (claims 2-3, and 8).
Conclusion
No claims are allowable.
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QIWEN SU-TOBON
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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