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 .
Response to Amendment/Status of Claims
Receipt of Arguments/Remarks filed on 10/07/2025 is acknowledged. Claims 1-33 are pending. Claims 20 and 27 were amended. Claims 1-19 and 28-33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 04/04/2025. Applicant elected “a folded RBD” as Species A in claim 20 in the reply filed on 04/04/2025.
Claims 20-27 are under examination.
Priority
This application is a 371 of PCT/US2020/018758 filed 02/19/2020, which claims benefit of 62/807,459 filed 02/19/2019 as reflected on the filing receipt dated 02/08/2022.
Applicant argues that support for instant claims 20-27 is found in the specification and claims of the ‘459 application, at least for example claims 17-22, [0003], [0004], and [0009].
This is not found persuasive. While the components of the instant protein system in a. of claim 20 have support in the ‘459 application, the examiner does not see support in the ‘459 application for a method for measuring phase behavior of natural or engineered multi-component condensates, comprising the steps recited in a., b., and c. of instant claim 20. While claim 17 of ‘459 recites a method for initiation and modulating material properties of physiological muti-component RNA granules comprising the steps of providing a protein system according to claim 1; and oligomerizing the folded RNA binding domain (RBD), disordered RBD, or folded non-RBD domain by exposing the light-sensitive receptor protein to at least one wavelength of light, there does not appear to be support for these steps combined with the measuring options recited in instant part c. There is no mention in ‘459 of determining if condensation or aggregation occurs.
Therefore, claims 20-27 receive the priority date of 02/19/2020, rather than 02/19/2019 of 62/807,459 as support for instant claims 20-27 was not found in 62/807,459.
Response to Arguments
Applicant’s arguments and amendments, see page 11, filed 10/07/2025, with respect to the objections to claim 20 have been fully considered and are persuasive, due to the amendments correcting the typos. The objection to claim 20 has been withdrawn.
Applicant’s arguments and amendments, see page 12, filed 10/07/2025, with respect to the 35 U.S.C. 112(b) rejection of claims 20-27 have been fully considered and are persuasive due to the amendment to step c. of claim 20 which make clear the options of measuring phase behavior. The 35 U.S.C. 112(b) rejection of claims 20-27 has been withdrawn.
Applicant’s arguments and amendments , see pages 14-17, filed 10/07/2025, with respect to the rejection(s) of claim(s) 20-25 and 27 under 35 U.S.C. 103 as unpatentable over Donnelly et al. (‘293) in view of Brangwynne et al. (‘977) and claim 26 as unpatentable over Donnelly in view of Brangwynne and further in view of Patel et al. have been fully considered and are persuasive due to the amendments to claims 20 and 27. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of the amendments to claims 20 and 27 regarding a case of obviousness. See the modified 103 rejections below.
Applicant’s arguments and amendments, see pages 17-18, filed 10/07/2025, with respect to the rejection(s) of claim(s) 20-27 under nonstatutory double patenting over claims 1,2,4-18,20-22,24,25 of co-pending Application no. 17/277,518 in view of Brangwynne have been fully considered and are persuasive due to the amendments to claims 20 and 27. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of the amendments to claims 20 and 27. See the modified double-patenting rejection below.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 20-25 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2018165293 (‘293), Published 13 September 2018, in view of US 20170355977 (‘977), Published 14 Dec 2017, cited on an IDS dated 08/18/2021, and US 20180251497 (‘497), Published 6 Sep 2018, cited on an IDS dated 03/09/2022.
Claim Interpretation: The instant specification discloses that the light-sensitive protein is fused to a folded RBD, and the folded RBD is an RNA recognition motif (RRM), a K homology (KH) domain, a Pumilio (PUM) domain, a zinc-finger domain, a DEAD box helicase domain, a double-stranded RNA-binding domain (dsRBD), an m6A RNA-binding domain (YTH domain), or a Cold shock domain (CSD) (paragraph 0016). Therefore, any of the above recited domains would be considered to be a folded RBD.
The instant specification also discloses the light-sensitive proteins or cognate partners can be any light-sensitive proteins or cognate partners known to those of skill in the art, including natural or engineered proteins, such as BLUF domains (such as bPAC), Phytochromes (such as Phy-PIF or BpbP1-PpsR2), Cryptochromes (such as LARIAT, LITE, OPTOSTIM, Cryptochrome 2 and CIB1), LOV domains (such as BACCS, LAD, LITEZ, iLID [LOV2-SsrA]/SspB, pDawn, and pDUSK)…In one preferred embodiment the first fusion protein uses a single LOV2-SsrA protein (paragraph 0058). Paragraph 0059 goes on to describe using the iLID system where LOV2-SsrA is a light-sensitive protein, and SspB is its cognate partner). Therefore, art that teaches any of these folded RBDs, light-sensitive proteins, and cognate partners of the light-sensitive proteins, reads on the instant claims.
In addition regarding claim 23, the examiner is interpreting a stress granule to be a cytoplasmic ribonucleoprotein granule (claim 23), based on paragraph 0006 of the instant specification.
Regarding claims 20 and 27, ‘293 teaches a method of screening for an agent that modulates protein aggregation, comprising the steps of introducing into a cell an expression vector encoding a chimeric polypeptide, comprising a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding for a low complexity domain from a neurodegenerative disease target protein, expressing the chimeric polypeptide, introducing the agent into culture media comprising the cell, inducing oligomerization of the chimeric polypeptide by stimulation with blue light and determining modulation of protein aggregation by the agent (page 2 lines 32-34, and page 3 lines 1-7). ‘293 teaches the low complexity domain from a neurodegenerative disease target protein may be TDP-43, alpha-synuclein, Tau, FUS (page 3, lines 18-19). ‘293 teaches that Fig. 3 shows light induced oligomerization and aggregation of proteins employing the NcVVD, NcVVDY50W or NcLOV photoreceptor, and the top panel shows a schematic of how a single acute stimulation with blue light (405-499 nm) induces the homodimerization of the LOV protein when fused to a protein of interest, and the bottom panel shows chronic stimulation with blue light promotes homooligomerization of NcVVD or LOV fusion proteins that contain a prion-like domain/LCD/IDD (page 4, lines 29-34- page 5, lines 1-2).
‘293 teaches quantifying phase separation or aggregation based on an amount of fluorescence within a first region and second region of the plurality of cells in which fluorescence recovery after photobleaching imaging was performed to assess dynamicity of optoTDP43 structures, and that lack of fluorescence recovery shows that light-induced optoTDP43 aggregates are non-dynamic, immobile granules reminiscent of aggregated structures (page 5, lines 20-23; Fig. 5E); Fig. 6A shows chronic stimulation paradigm and Fig. 6B shows representative images of a-synuclein clustering with light over time along with quantification of clustering in Fig. 6C (page 6, lines 18-20).
Regarding claim 21, ‘293 teaches the constructs of fusion proteins were fused to a fluorescent protein, mCherry to visualize the proteins in live cells (page 4, lines 23-24).
While ‘293 teaches a screening method using a chimeric polypeptide comprising a first nucleotide sequence encoding a light-induced oligomerization domain (light-sensitive protein) and a second nucleotide sequence encoding for a low complexity domain from a neurodegenerative disease target protein, ‘293 does not teach using a protein system comprising an optoprotein comprising a cognate partner of a light-sensitive protein fused to a second region comprising one or more folded RNA binding domains, and does not teach that the chimeric polypeptide comprising a light-induced oligomerization domain is a core protein or that it is fused to one or more folded RNA binding domains, and that the second region of the core protein is adapted to self-assemble. ‘293 does not teach that the protein system is located outside of a living or dead cell, that oligomerization drives gelation of a cytoplasmic ribonucleoprotein granule, or the protein system is in a well in a multi-well array/plate.
However, before the effective filing date, ‘977 teaches protein constructs with the ability to induce and control reversible liquid-liquid phase separation in living cells, and that the location within the phase diagram can be used to dictate the material state of phase-separated IDR clusters, ranging from dynamic liquid droplets to arrested but reversible gels which can over time mature into irreversible aggregates, and the protein constructs comprise light sensitive proteins fused to a low complexity sequence (LCS) or intrinsically disordered protein region (IDR) (paragraph 0008). ‘977 teaches the protein construct comprises a first segment comprising a gene encoding at least one protein sensitive to light, and a second segment fused to the first segment comprising a synthetic or natural nucleic acid binding domain, which is selected from an RNA recognition motif, zinc-finger binding domains, Pumilio or YT521-B homology (YTH). See above claim interpretation section where these same domains are considered to be folded RBDs according to the instant specification (paragraph 0016).
‘977 teaches at least three protein construct system configurations are envisioned that utilize multiple, different protein constructs (paragraph 0062), and in the second configuration at least two types of constructs are used, having different light sensitive regions, and the two types of constructs each comprising at least a portion of one of a pair of proteins, such as Cry2-CIB, PhyB-PIF, or iLID-SspB (0063). See claim interpretation above where the instant specification stated these to be light-sensitive proteins and their cognate partners.
‘977 teaches FUS is found in stress granules which is a type of membrane-less body whose assembly depends on PTMs and protein concentration and has been suggested to assemble by regulated intracellular phase separation (paragraph 0038).
‘977 teaches dynamically tuning protein interactions with light achieves high degree of control over intracellular phase space, which can be exploited to study the phase diagram of FUS-mediated assemblies within living cells, and varying the degree of quenching depth leads to clusters spanning different material states, ranging from liquid droplets to gels (paragraph 0071). ‘977 teaches deep quenching results in formation of gels which exhibit minimal molecular dynamics and highly irregular aggregate-like morphologies (paragraph 0071). ‘977 teaches that increasing the strength or effective valency of molecule self-association (e.g. through light activation) can lead to liquid-liquid phase separation, or for higher supersaturation can result in gelation, and it is known that membrane-less organelles can exhibit at least partially solid-like properties (paragraph 0072). ‘977 teaches that large variations in the immobile fraction of stress granule proteins are often measured in FRAP experiments, and in some cases stress granules begin to resemble irregularly shaped gels. ‘977 teaches the ability to tune material states by moving within the phase diagram could be exploited by cells, since highly dynamic liquid-like states may be useful as microreactors, while gel-like structures provide an ideal storage environment (paragraph 0072).
‘977 teaches the protein construct is expressed in cells which are then lysed (paragraph 0073), and therefore teaches the protein system is located outside of a dead cell.
‘977 teaches the constructs of mCherry-labeled Cry2 PHR are introduced into living cells, and that 293T and NIH 3T3 cells are plated in 6-well dishes, and NIH 3T3 cells plated in the 6-well dishes were infected by adding filtered viral supernatant to the cell medium, the cells were then induced to cluster with blue light, and that fusing the N-terminal IDR of FUS to Cry2 WT leads to rapid blue-light dependent cluster assembly in most cells (paragraph 0040-0041), and therefore teaches the protein system is present in a well of a multi-well array or plate, and adding chemical agents to the well.
Additionally, ‘497 teaches a platform for reversibly and non-reversibly generating liquid droplets, gels, or protein aggregates inside and outside cells by using nucleation cores, which may be controlled by light. In the present invention, systems and methods are provided for a system of protein constructs which may utilize a photo-activatable or photo-deactivatable interaction between protein partners to control the recruitment of intrinsically disordered proteins on self-assembling protein cores (see, e.g., FIG. 4). Light may be used to trigger the assembly or possibly disassembly of an interactive layer, where one of the protein pairs is fused to a full length or truncated low complexity or intrinsically-disordered protein (see, e.g., FIG. 5). In other systems, a self-assembling protein core is fused to a full length or truncated low complexity or intrinsically disordered protein (paragraph 0008). ‘497 teaches among the many different possibilities contemplated, the self-assembling protein subunit could be a ferritin heavy chain, and the intrinsically disordered region (IDR) can be the N terminal domain of FUS protein. Photo-inducible reversible heterodimerization between the self-assembling and IDR units could utilize the engineered blue light activatable iLID protein and its cognate partner, sspB (paragraph 0009). FIGS. 1A and 1B depict generalized embodiments of the disclosed platform, which generally comprises two types of protein constructs (12, 14) (paragraph 0024).
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‘497 teaches the first construct (12) comprises at least one light sensitive receptor component (20) fused to a self-assembling oligomeric protein subunit (30). The light sensitive receptor component (20) may comprise one or more similar or different proteins responsive to at least one wavelength of light, preferably a wavelength of light in the near UV, visible or infra-red regions, which are from about 350 nm to about 800 nm. In preferred embodiments, the light sensitive protein is the engineered protein iLID, which consist of a modified LOV2 domain fused at its C terminus to an ssrA peptide. However, other light sensitive proteins may also be utilized, including Cry2, PhyB or a LOV2 domain fused to a signaling peptide other than ssrA. The self-assembling protein subunit (30) can be any protein that self-assembles, including but not limited to ferritin light chains, ferritin heavy chains, glutamine synthetase, and viral capsid structure proteins. One preferred embodiment utilizes ferritin heavy chain subunits, which are capable of self-assembly into a 24 mer complex with a spherical shell structure (paragraph 0025).
‘497 teaches the second type of construct (14) comprises at least one cognate partner (40) of the light sensitive receptor component (20), fused to a full length or truncated low complexity sequence (LCS) or IDR (60) (paragraph 0027). The cognate partner (40) is any appropriate cognate of the light sensitive receptor component (20), which may include but is not limited to ssrB, Zdk, CIB, or PIF for LOV2-ssrA, LOV2, Cry2, or PhyB respectively. In preferred embodiments, the second protein construct comprises an IDP (60), which include but not limited to full length or truncated forms of FUS, DDX4, and hnRNPA1 (paragraph 0028). ‘497 teaches an example of recruitment of endogenous FUS protein by core based droplets can be seen by utilizing a first construct comprising ferritin fused to two iLID-ssrA domains, and a second construct comprising FUSn fused to mCherry and sspB
‘497 also recites a construct system, comprising: a first construct comprising at least one self-assembling protein subunit fused to at least one light-sensitive receptor protein; and a second construct comprising a cognate partner of the light-sensitive receptor protein fused to a full length or truncated low complexity or intrinsically-disordered protein region (claim 1); The construct system according to claim 1, wherein the self-assembling protein subunit is ferritin (claim 2); The construct system according to claim 4, wherein the engineered protein is iLID (claim 5); The construct system according to claim 1, wherein the cognate partner is sspB (claim 7).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to substitute the chimeric protein used in the method of ‘293 with the protein construct systems of ‘977 and ‘497 to arrive at the instant claimed method that provides the protein system comprising one or more optoproteins and a core protein as instantly claimed with a reasonable expectation of success. There would be a reasonable expectation of success as this amounts to substituting the known chimeric protein of ‘293 with known protein constructs of ‘977 and ‘497 to arrive at predictable results. In addition, ‘293, ‘977 and ‘497 are in the same field of chimeric proteins/fusion proteins containing light-sensitive domains, including applying light to the chimeric/fusion protein, and both ‘293 and ‘977 relate to determining phase separation or aggregation, and using a phase diagram. In addition, both ‘977 and ‘497 teach two types of protein constructs are used. ‘977 teaches a protein construct comprises a first segment comprising a gene encoding at least one protein sensitive to light, and a second segment fused to the first segment comprising a synthetic or natural nucleic acid binding domain and that at least two types of constructs are used, having different light sensitive regions, and the two types of constructs each comprising at least a portion of one of a pair of proteins, such as Cry2-CIB, PhyB-PIF, or iLID-SspB (0063) which are light-sensitive proteins and their cognate partners. ‘497 teaches the first construct comprises at least one light sensitive receptor component (20) fused to a self-assembling oligomeric protein subunit and a second type of construct comprises at least one cognate partner (40) of the light sensitive receptor component (20), fused to a full length or truncated low complexity sequence (LCS) or IDR (60) (paragraph 0027). One of ordinary skill in the art would have been motivated to substitute the chimeric protein used in the method of ‘293 with the protein construct systems of ‘977 and ‘497 because ‘977 teaches protein constructs with these domains with the ability to induce and control reversible liquid-liquid phase separation in living cells and because ‘497 teaches a platform for reversibly and non-reversibly generating liquid droplets, gels, or protein aggregates inside and outside cells by using nucleation cores, which may be controlled by light, and systems and methods are provided for a system of protein constructs which may utilize a photo-activatable or photo-deactivatable interaction between protein partners to control the recruitment of intrinsically disordered proteins on self-assembling protein cores. Regarding step c. of claim 20, ‘293 teaches quantifying phase separation or aggregation and ‘977 teaches that the location within the phase diagram can be used to dictate the material state of phase-separated IDR clusters, and that dynamically tuning protein interactions with light achieves high degree of control over intracellular phase space, which can be exploited to study the phase diagram of FUS-mediated assemblies within living cells. Accordingly, the limitations of claims 20-21 are made obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date to substitute the chimeric protein used in the method of ‘293 with the protein construct systems of ‘977 and ‘497 and to modify the method of ‘293, with the teachings of ‘977 regarding the optoprotein system being located outside of a dead cell, or the optoprotein system to be in a well in a multi-well array/plate with a reasonable expectation of success. There would be a reasonable expectation of success, because ‘293, ‘977 and ‘497 are in the same field of chimeric proteins or fusion proteins containing light-sensitive domains, including applying light to the chimeric/fusion protein, and determining phase separation or aggregation, and using a phase diagram. One of ordinary skill in the art would have been motivated to provide the protein system outside of a dead cell, because ‘977 teaches the protein construct is expressed in cells which are then lysed, and therefore teaches the protein system is located outside of a dead cell. One of ordinary skill in the art would have been motivated to provide the protein system in a well in a multi-well array/plate and providing one or more chemical agents to the well and determining the impact thereof, because ‘977 teaches that constructs of mCherry-labeled Cry2 PHR are introduced into living cells, and that 293T and NIH 3T3 cells are plated in 6-well dishes, and NIH 3T3 cells plated in the 6-well dishes were infected by adding filtered viral supernatant to the cell medium, the cells were then induced to cluster with blue light, and that fusing the N-terminal IDR of FUS to Cry2 WT leads to rapid blue-light dependent cluster assembly in most cells (paragraph 0040-0041). Accordingly, the limitations of instant claims 22,24,25 and 27 are obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date to substitute the chimeric protein used in the method of ‘293 with the protein construct systems of ‘977 and ‘497 and to modify the method of ‘293 in view of the teachings of ‘977 with a reasonable expectation of success, as ‘977 teaches deep quenching results in formation of gels which exhibit minimal molecular dynamics and highly irregular aggregate-like morphologies (paragraph 0071), increasing the strength or effective valency of molecule self-association (e.g. through light activation) can lead to liquid-liquid phase separation, or for higher supersaturation can result in gelation, that large variations in the immobile fraction of stress granule proteins are often measured in FRAP experiments, and in some cases stress granules begin to resemble irregularly shaped gels. One of ordinary skill in the art would have been motivated to do so because ‘977 teaches the ability to tune material states by moving within the phase diagram could be exploited by cells, since highly dynamic liquid-like states may be useful as microreactors, while gel-like structures provide an ideal storage environment (paragraph 0072), and would make obvious the limitations of claim 23.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claims 26 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over ‘293 in view of ‘977 and ‘497 as applied to claims 20-25 and 27 above, and further in view of Patel et al. (Cell 162, 1066-1077, 27 August 2015), cited on an IDS.
Claim 27 is added to this rejection based on the amendment to claim 27 to include a step of performing a genetic screen based on gene knockdown, a genetic screen based on gene upregulation in order to reject an additional species.
The teachings of ‘293, ‘977 and ‘497 as applicable to claims 20-25 and 27 are described above.
‘293, ‘977 and ‘497 do not teach utilizing a genetic screen based on gene knockdown or gene upregulation, or performing a genetic screen based on gene knockdown, or gene upregulation.
However, before the effective filing date, Patel et al. teach many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases as they are prone to aggregate, and study FUS which is a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS (Summary, page 1066). FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress (Summary, page 1066). Patel et al. teach FUS is an RNA-binding protein, and mutations in which are associated with ALS and frontotemporal lobar degeneration (page 1066). Patel et al. teach that various studies have implicated FUS in formation of stress-inducible compartments such as DNA damage sites and stress granules, and have used overexpression plasmids to study subcellular localization of FUS (Page 1067).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the method of ’293, ‘977 and ‘497 to further include utilizing or performing a genetic screen based on gene upregulation in order to study the effect that overexpression of FUS would have on subcellular localization as taught by Patel et al. with a reasonable expectation of success. There would be a reasonable expectation of success because ‘293, ‘977, and Patel et al. all describe FUS as found in stress granules and is associated with neurodegenerative diseases and whose assembly is based on phase separation and ‘497 discusses recruitment of FUS protein by core-based droplets using the first and second protein constructs. One of ordinary skill in the art would have been motivated to use a genetic screen based on gene upregulation in the method in order to study the effect that overexpression of FUS would have on subcellular localization as well as stress granule formation.
Accordingly, the limitations of claims 26 and 27 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
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 20-27 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1,2,4-18,20-22,24 and 25 of copending Application No. 17/277,518 (‘518) in view of US 20170355977 (‘977), Published 14 Dec 2017 and US 20180251497 (‘497), Published 6 Sep 2018.
Application ‘518 claims 1,2,4-18,20-22,24 and 25 recite a high-throughput method for mapping or screening intracellular interactions comprising steps a)-h). Step a) includes providing a plurality of cells each expressing a phase separation or aggregation system capable of being controlled by at least one wavelength and comprising (i) a target protein and (ii) a fluorescent protein or fluorophore; step b) is placing at least one of the plurality of cells in a well; step c) is introducing at least one chemical or biological agent to the well; step d) is irradiating the well with at least one wavelength of light and allowing the phase separation or aggregation system to form a condensate; step f) including quantifying phase separation or aggregation and step g) including generating a phase diagram utilizing the quantified phase separation or aggregation. Claim 4 recites the phase separation or aggregation system comprises a first and second construct, with the first comprising a Cas9 fused or attached to two or more repeating sequences, each including a receptor protein sensitive to the at least one wavelength of light, and a second construct comprising a cognate partner of the receptor protein fused to the fluorescent protein and at least one gene regulatory protein having a low complexity or intrinsically disordered protein region or a folded protein that promotes at least one of self-interactions, a network of heterotrophic interactions, or phase separation. Claim 7 recites the chemical or biological agent is a component of a gene knockout or knockdown screening system, and claim 8 recites the screening system is selected from TALEN, shRNA, siRNA and CRISPR-KO.
Application ‘518 does not recite the phase separation or aggregation system used in the method is composed of the components of the protein system recited in amended claim 20, or that oligomerization drives gelation of a cytoplasmic RNP granule.
However, before the effective filing date, ‘977 teaches protein constructs with the ability to induce and control reversible liquid-liquid phase separation in living cells, and that the location within the phase diagram can be used to dictate the material state of phase-separated IDR clusters, ranging from dynamic liquid droplets to arrested but reversible gels which can over time mature into irreversible aggregates, and the protein constructs comprise light sensitive proteins fused to a low complexity sequence (LCS) or intrinsically disordered protein region (IDR) (paragraph 0008). ‘977 teaches the protein construct comprises a first segment comprising a gene encoding at least one protein sensitive to light, and a second segment fused to the first segment comprising a synthetic or natural nucleic acid binding domain, which is selected from an RNA recognition motif, zinc-finger binding domains, Pumilio or YT521-B homology (YTH). (See claim interpretation in the 103 rejection above, where these same domains are considered to be folded RBDs according to the instant specification (paragraph 0016)).
‘977 teaches FUS is found in stress granules which is a type of membrane-less body whose assembly depends on PTMs and protein concentration and has been suggested to assemble by regulated intracellular phase separation (paragraph 0038). ‘977 teaches dynamically tuning protein interactions with light achieves high degree of control over intracellular phase space, which can be exploited to study the phase diagram of FUS-mediated assemblies within living cells, and varying the degree of quenching depth leads to clusters spanning different material states, ranging from liquid droplets to gels (paragraph 0071). ‘977 teaches deep quenching results in formation of gels which exhibit minimal molecular dynamics and highly irregular aggregate-like morphologies (paragraph 0071). ‘977 teaches that increasing the strength or effective valency of molecule self-association (e.g. through light activation) can lead to liquid-liquid phase separation, or for higher supersaturation can result in gelation, and it is known that membrane-less organelles can exhibit at least partially solid-like properties (paragraph 0072). ‘977 teaches that large variations in the immobile fraction of stress granule proteins are often measured in FRAP experiments, and in some cases stress granules begin to resemble irregularly shaped gels. ‘977 teaches the ability to tune material states by moving within the phase diagram could be exploited by cells, since highly dynamic liquid-like states may be useful as microreactors, while gel-like structures provide an ideal storage environment (paragraph 0072).
‘977 teaches the protein construct is expressed in cells which are then lysed (paragraph 0072), and therefore teaches the protein system is located outside of a dead cell.
Additionally, ‘497 teaches a platform for reversibly and non-reversibly generating liquid droplets, gels, or protein aggregates inside and outside cells by using nucleation cores, which may be controlled by light. In the present invention, systems and methods are provided for a system of protein constructs which may utilize a photo-activatable or photo-deactivatable interaction between protein partners to control the recruitment of intrinsically disordered proteins on self-assembling protein cores (see, e.g., FIG. 4). Light may be used to trigger the assembly or possibly disassembly of an interactive layer, where one of the protein pairs is fused to a full length or truncated low complexity or intrinsically-disordered protein (see, e.g., FIG. 5). In other systems, a self-assembling protein core is fused to a full length or truncated low complexity or intrinsically disordered protein (paragraph 0008). ‘497 teaches among the many different possibilities contemplated, the self-assembling protein subunit could be a ferritin heavy chain, and the intrinsically disordered region (IDR) can be the N terminal domain of FUS protein. Photo-inducible reversible heterodimerization between the self-assembling and IDR units could utilize the engineered blue light activatable iLID protein and its cognate partner, sspB (paragraph 0009). FIGS. 1A and 1B depict generalized embodiments of the disclosed platform, which generally comprises two types of protein constructs (12, 14) (paragraph 0024).
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‘497 teaches the first construct (12) comprises at least one light sensitive receptor component (20) fused to a self-assembling oligomeric protein subunit (30). The light sensitive receptor component (20) may comprise one or more similar or different proteins responsive to at least one wavelength of light, preferably a wavelength of light in the near UV, visible or infra-red regions, which are from about 350 nm to about 800 nm. In preferred embodiments, the light sensitive protein is the engineered protein iLID, which consist of a modified LOV2 domain fused at its C terminus to an ssrA peptide. However, other light sensitive proteins may also be utilized, including Cry2, PhyB or a LOV2 domain fused to a signaling peptide other than ssrA. The self-assembling protein subunit (30) can be any protein that self-assembles, including but not limited to ferritin light chains, ferritin heavy chains, glutamine synthetase, and viral capsid structure proteins. One preferred embodiment utilizes ferritin heavy chain subunits, which are capable of self-assembly into a 24 mer complex with a spherical shell structure (paragraph 0025).
‘497 teaches the second type of construct (14) comprises at least one cognate partner (40) of the light sensitive receptor component (20), fused to a full length or truncated low complexity sequence (LCS) or IDR (60) (paragraph 0027). The cognate partner (40) is any appropriate cognate of the light sensitive receptor component (20), which may include but is not limited to ssrB, Zdk, CIB, or PIF for LOV2-ssrA, LOV2, Cry2, or PhyB respectively. In preferred embodiments, the second protein construct comprises an IDP (60), which include but not limited to full length or truncated forms of FUS, DDX4, and hnRNPA1 (paragraph 0028). ‘497 teaches an example of recruitment of endogenous FUS protein by core based droplets can be seen by utilizing a first construct comprising ferritin fused to two iLID-ssrA domains, and a second construct comprising FUSn fused to mCherry and sspB
‘497 also recites a construct system, comprising: a first construct comprising at least one self-assembling protein subunit fused to at least one light-sensitive receptor protein; and a second construct comprising a cognate partner of the light-sensitive receptor protein fused to a full length or truncated low complexity or intrinsically-disordered protein region (claim 1); The construct system according to claim 1, wherein the self-assembling protein subunit is ferritin (claim 2); The construct system according to claim 4, wherein the engineered protein is iLID (claim 5); The construct system according to claim 1, wherein the cognate partner is sspB (claim 7).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to substitute the phase separation or aggregation system capable of being controlled by light used in the method of ‘518, the protein construct systems of ‘977 and ‘497 to arrive at the instant claimed method that provides the protein system comprising one or more optoproteins and a core protein as instantly claimed with a reasonable expectation of success. There would be a reasonable expectation of success as this amounts to substituting the phase separation or aggregation system comprising the first and second construct of ‘518 with known protein constructs of ‘977 and ‘497 to obtain predictable results. In addition, ‘977 and ‘497 are in the same field of chimeric proteins/fusion proteins containing light-sensitive domains, including applying light to the chimeric/fusion protein, and ‘977 relates to determining phase separation or aggregation, and using a phase diagram. In addition, both ‘977 and ‘497 also teach two types of protein constructs are used. ‘977 teaches a protein construct comprises a first segment comprising a gene encoding at least one protein sensitive to light, and a second segment fused to the first segment comprising a synthetic or natural nucleic acid binding domain and that at least two types of constructs are used, having different light sensitive regions, and the two types of constructs each comprising at least a portion of one of a pair of proteins, such as Cry2-CIB, PhyB-PIF, or iLID-SspB (0063) which are light-sensitive proteins and their cognate partners. ‘497 teaches the first construct comprises at least one light sensitive receptor component (20) fused to a self-assembling oligomeric protein subunit and a second type of construct comprises at least one cognate partner (40) of the light sensitive receptor component (20), fused to a full length or truncated low complexity sequence (LCS) or IDR (60) (paragraph 0027). One of ordinary skill in the art would have been motivated to substitute the phase separation or aggregation system capable of being controlled by light used in the method of ‘518 with the protein construct systems of ‘977 and ‘497 because ‘977 teaches protein constructs with these domains with the ability to induce and control reversible liquid-liquid phase separation in living cells. In addition, ‘497 teaches a platform for reversibly and non-reversibly generating liquid droplets, gels, or protein aggregates inside and outside cells by using nucleation cores, which may be controlled by light, and systems and methods are provided for a system of protein constructs which may utilize a photo-activatable or photo-deactivatable interaction between protein partners to control the recruitment of intrinsically disordered proteins on self-assembling protein cores. ‘977 teaches that the location within the phase diagram can be used to dictate the material state of phase-separated IDR clusters, and that dynamically tuning protein interactions with light achieves high degree of control over intracellular phase space, which can be exploited to study the phase diagram of FUS-mediated assemblies within living cells.
It would have been obvious to one of ordinary skill in the art before the effective filing date that the method of ‘518 would include the step of oligomerization that would drive gelation of a cytoplasmic ribonucleoprotein (RNP) granule in view of the teachings of ‘977 which teach deep quenching results in formation of gels which exhibit minimal molecular dynamics and highly irregular aggregate-like morphologies (paragraph 0071), that large variations in the immobile fraction of stress granule proteins are often measured in FRAP experiments, and in some cases stress granules begin to resemble irregularly shaped gels. One of ordinary skill in the art would have been motivated to do so because ‘977 teaches the ability to tune material states by moving within the phase diagram could be exploited by cells, since highly dynamic liquid-like states may be useful as microreactors, while gel-like structures provide an ideal storage environment (paragraph 0072).
Accordingly, the limitations of claims 20-27 would have been prima facie obvious.
This is a provisional nonstatutory double patenting rejection.
Conclusion
Claims 20-27 are rejected.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636