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 .
Application Status
Applicant’s preliminary amendment filed February 6, 2023 is acknowledged. Claims 1-2, 4-5, 7-10, 14, 18-20, 22, 24-30, 34-36, 41, 47, 49, 53, 59 and 100 are pending and under examination.
The petition to accept color drawings filed May 15, 2023 was granted.
Specification
The use of the term SpyCatcher, SpyTag, HaloTag, SNAP-tag, SLIP-tag, Alexa Flour, GeneJet, Teflon, Opti-MEM, and TransIT, which are trade names or marks used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
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 5 and 20 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 5 is indefinite for two reasons.
First, claim 5 contains the trademark/trade names SpyCatcher and SpyTag. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe two proteinaceous domains that can self-ligate and, accordingly, the identification/description is indefinite.
Second, claim 5 recites “wherein the protein assembly monomers are selected from the group consisting of…” and then recites 10 different polymers including microtubules, prions, and crystals of the kinase PAK4. Claim 5 is confusing because all but one of the species listed in the Markush group for the “assembly monomers” are actually polymers and not monomers.
To remedy the indefiniteness, it is suggested that SpyCatcher and SpyTag be replaced with generic terminology if possible and for the claim to recite “wherein the protein assembly monomers are monomers capable of forming polymers selected from the group consisting of endogenous microtubules…”
It is noted that claim 7 is not included in this §112(b) rejection, because it is clear that the monomers are iPAK4, which according to the Specification, is a monomer.
Claim 20 recites “wherein the visualizable tag comprises… a small molecule tag that binds fluorescent dyes… a nucleic acid tag that binds fluorescent dyes”. Claim 20 depends from claim 19, which recites “where the protein assembly monomer encoded by the first polynucleotide is fused to a visualizable tag”. Therefore, the visualizable tag fused to the monomer must be genetically encoded. The Specification does not provide examples of small molecules or nucleic acids that can bind fluorescent dyes ([0066]). The only examples of molecules that can bind fluorescent dyes are peptides and proteins, which can be encoded by a polynucleotide. Small molecules are not capable of being encoded by polynucleotides. Although nucleic acids are themselves genetic molecules, it is unknown how a nucleic acid fused to a protein can be genetically encoded. As such, it is confusing how a small molecule and a nucleic acid can be comprised in a tag that must be encoded by a polynucleotide.
To remedy the indefiniteness, it is suggested that “a small molecule tag that binds fluorescent dyes” and “a nucleic acid tag that binds fluorescent dyes” be removed from the claim.
Claim Interpretation
Claim 1 recites first and second polynucleotides encoding a first and second protein assembly monomer. The Specification teaches “protein assembly can be composed of the same monomer. For example, a protein assembly may be composed of multiple monomers that are the same, except for various modifications as described herein. In some embodiments, a protein assembly is composed of multiple different monomers.” ([0055]). Claim 1 also recites the first and second polynucleotides comprise a first and second promoter, respectively, for driving expression of the protein assembly monomers. The Specification states “In some embodiments, the transcripts produced by the first and the second polynucleotide joined together as a single expression vector are produced by alternative splicing of a single parent transcript driven by the same promoter.” ([0095]). The broadest reasonable interpretation of the first and second polynucleotides is that they can encode the same or different protein assembly monomers, such as the same assembly monomer fused to different fluorescent proteins. Additionally, the first and second promoters can be the same or different.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 100 is rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception without significantly more. The claim recites a pair of polynucleotides, which is a natural product. However, the claims do not include elements, when considered separately and in combination, that are sufficient to amount to significantly more than the judicial exceptions as outlined below.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES
Claim 100 is directed to a pair of polynucleotides. Thus, the claims are directed to a statutory category (e.g., a product).
Step 2A, Prong One - Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES
Natural phenomena have been identified by the courts by way of example, including natural products and natural correlations. For natural products, products that are not “markedly different” than their naturally occurring counterpart are judicial exceptions. See MPEP 2016.04((b). The courts have identified “isolated DNA” as one such natural product that may not be markedly different from their counterpart, DNA in cells. MPEP 2106.04(c) outlines the markedly different analysis which is applied to the claimed product as follows.
The claim recites a pair of polynucleotides and 5 options for the pair. The first option recites “wherein the first polynucleotide encodes a first protein assembly monomer under the control of a first promoter and a second protein encoding a second protein assembly monomer under control of a second promoter. The Specification indicates that protein assembly monomers include cytoskeletal elements such as microtubules ([0061]). Microtubules are well known in the art to be composed of genetically-encoded b-tubulin and a-tubulin monomers that polymerize to form microtubules. See e.g., Desai and Mitchison, Annu Rev. Cell Dev. Biol. (1997), 13: 83-117. Therefore, the genes that code for b-tubulin and a-tubulin, TUBB and TUBA1A, respectively, are encompassed by a polynucleotide encoding a first protein assembly monomer and a polynucleotide encoding a second protein assembly monomer. Genbank teaches that the human TUBB and TUB1A genes are on chromosomes 6 and 12, respectively and are flanked on the 5’ end by upstream transcriptional regulation sequence. (Gene ID: 7846, TUBA1A tubulin alpha 1a [Homo sapiens (human)], https://www.ncbi.nlm.nih.gov/gene/7846, [retrieved December 1, 2025] and Gene ID: 203068, TUBA1A tubulin beta class I [Homo sapiens (human)], https://www.ncbi.nlm.nih.gov/gene/203068 [retrieved December 1, 2025]). Thus, the closest naturally occurring counterpart to the claimed the pair of polynucleotides are human chromosomes 6 and 12. The claimed pair of polynucleotides in option (i) is not markedly different than human chromosomes 6 and 12, and therefore constitutes a judicial exception
Step 2A, Prong Two - Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. The phrase "integration into a practical application" requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception. In this case, the claim does not recite any additional elements that integrates the pair of polynucleotides into a practical application.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO
The Supreme Court has identified a number of considerations for determining whether a claim with additional elements amounts to "significantly more" than the judicial exception(s) itself. The claim as a whole is evaluated as to whether it amounts to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements, adds an inventive concept to the claim (MPEP 2106.05). However, the claim does not recite any additional elements and therefore the claim does not amount to "significantly more" than the pair of polynucleotides.
Claim Rejections - 35 USC § 102 – Linghu
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2, 4-5, 8, 14, 19, 41, 47, 49, 53 and 100 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Linghu (Linghu et al., Cell (2020), 183: 1682-1698). Claim 47 is evidenced by Barnett (Barnett et al., PLoS One (2017), 12(2): e0170934).
Regarding claims 1 and 8, Linghu teaches expressing two self-assembling peptide sets in a cell (i.e. a first protein assembly and a second protein assembly) (Fig 1B). Linghu teaches that each self-assembling peptide set comprises a self-assembly peptide monomer (i.e., a first and second protein assembly monomer) fused to an epitope and fused to a fluorescent reporter (Fig 1B). Linghu teaches transfecting cells with plasmids encoding the self-assembling peptide sets (page e6, ¶3-4). Linghu teaches each of the self-assembly peptide set reporters was driven by the UBC promoter (i.e., an activity-independent constitutive promoter) (Figs 1, 6 and 7). Thus, each of the polynucleotides encodes a protein assembly monomer under the control of a promoter. Linghu teaches expressing three different self-assembling peptide sets in neurons (Fig 3). Linghu teaches monitoring the change in size and fluorescence of puncta formed by the self-assembling peptide sets upon treatment with forskolin (i.e., a cellular event leads to a visualizable change of the first and second protein monomer in the protein assembly) (Fig 3).
Regarding claim 2, Linghu teaches the cellular event is the treatment with forskolin (i.e., concentration of a small molecule) (Fig 3).
Regarding claims 4-5, Linghu teaches the self-assembling peptides HexCoil-Ala and I3-O1 spontaneously form homo-tetramers and polyhedras (i.e., lattice-forming peptides), respectively (¶ spanning pages 1684-1686).
Regarding claim 14, Linghu teaches monitoring the change in puncta formation in multiple cells at once (Fig 4).
Regarding claim 19, Linghu teaches each of the self-assembling peptide reporters comprising a fluorescent reporter (i.e., a visualizable tag) (Figs 1, 3).
Regarding claim 41, the GFP based sensors in Linghu’s method are genetically-encoded protein-based visualizable tags (page 14, ¶4), and are therefore inherently susceptible to cleavage by a protease.
Regarding claim 47, Linghu teaches the fluorescent sensor GCaMP6f is a Ca+ indicator that increases fluorescence upon Ca+ binding (i.e., the visualizable protein is visualizable only after being modified due to the cellular event) (page 1684, ¶1), which increased upon incubation with forskolin (Fig 4).
Regarding claim 49, Linghu is silent as to the mechanism of GCaMP6f fluorescence. Barnett teaches that upon Ca+ binding, GCaMP6f has a change in protonation state (i.e., a post-translational modification) (Abstract). Therefore, the self-assembling peptide reporter inherently comprised a visualizable tag that becomes visible due to a post-translational modification of the GCaMP6f reporter when it binds Ca+, which was due to the cellular event of forskolin signaling.
Regarding claim 53, Linghu teaches self-assembling peptide reporter comprising GCaMP6f, which binds Ca+ binding that is produced upon forskolin addition (i.e., the protein assembly monomer is fused to a binding-moiety that binds a molecule associated with the occurrence of the cellular event being profiled) (Fig 4; page 1684, ¶1).
Regarding claim 100, options (i) and (ii), Linghu teaches a plasmid encoding for the expression of S1-GCaMP6f (i.e., a first polynucleotide encoding a first protein assembly monomer fused to a visualizable tag under control of a first promoter) and a plasmid for the expression of S2-cAMPr (i.e., a second polynucleotide encoding a second protein assembly monomer fused to a visualizable tag under control of a second promoter) (Fig 3; page e2, Table).
Claim Rejections - 35 USC § 102 - Li
Claims 1-2, 4-5, 7, 14, 19, 41, 53 and 100 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Li (Li et al., Nano Letters (2019), 19: 6955-6963).
Regarding claim 1, 5 and 7, Li teaches expressing three proteins in a cell, wherein each protein comprises the Pak4 catalytic domain (Pak4cat) fused to the iBox domain from Inka1 (iBox), which is termed inka-PAK4 (Fig 1). Li teaches inka-PAK4 forms a crystal lattice in cells (i.e., inka-Pak4 is a protein assembly monomer) (Fig 1a). Li teaches a first protein comprises only inka-Pak4, a second protein comprises GFP fused to inka-Pak4 and termed GFP-Pak4, and the third protein comprises the iron-binding domain ferratin fused to inka-Pak4 and termed ftn-PAK4 (Fig 1a-b). Li teaches co-transfecting cells with plasmids encoding the three proteins (i.e., a first, second and third polynucleotide) (page 6961, ¶5). Because Li teaches each of the three proteins is expressed in cells (Fig 1e), the plasmids encoding the three iBoxPAK4cat-based proteins must have also comprised a promoter for driving the expression of the proteins. Li teaches inka-PAK4, GFP-PAK4, and ftn-PAK4 form a crystal lattice in cells (Fig 1e). Li teaches monitoring the location of the PAK4 crystals in cells upon applying a magnetic field to the cells (i.e., a cellular event) (Fig 4b). Li also teaches providing iron to cells (i.e., a cellular event) and monitoring PAK4 crystal length (¶ spanning pages 6960-6961).
Regarding claim 2, Li teaches providing iron to cells (i.e., a cellular event comprising change in the concentration of a small molecule) and monitoring PAK4 crystal length (¶ spanning pages 6960-6961).
Regarding claim 4, Li teaches the inka-Pak4-based proteins spontaneously assemble in cells (Fig 1; page 6959, ¶2).
Regarding claim 14, Li teaches monitoring multiple cells at once (Fig 4b).
Regarding claim 19, Li teaches one of the proteins is GFP-inka-PAK4 (i.e., the first protein assembly monomer is fused to a visualizable tag) (Fig 1).
Regarding claim 41, the GFP in Li’s GFP-inka-PAK4 is a genetically-encoded protein-based visualizable tag (page 14, ¶4), and is therefore inherently susceptible to cleavage by a protease.
Regarding claim 53, Li teaches ftn-inka-PAK4 can bind iron (i.e., the second assembly monomer is fused to a binding moiety that binds Fe, which is associated with the occurrence of the cellular event) (Figs 1, 4b; page 6956, ¶3).
Regarding claim 100, option (i) and (v), Li teaches a plasmid encoding for the expression of GFP-inka-PAK4 (i.e., a first polynucleotide encoding a first protein assembly monomer fused to a visualizable tag under control of a first promoter) and a plasmid for the expression of ftn-inka-PAK4 (i.e., a second polynucleotide encoding a second protein assembly monomer fused to a Fe-binding moiety under control of a second promoter) (Fig 3; page e2, Table).
Claim Rejections - 35 USC § 102 - Kahn
Claims 1-2, 4-5, 8, 18-19, 36, 41, 47, 49 and 100 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kahn (Kahn et al., Nature Communications (2018), 9:2773, pages 1-17). Claim 8 is evidenced by Hoogenraad (Hoogenraad et al., Nat. Neurosci. (2005): 8, 906–915). Claims 18 and 49 are evidenced by Henderson (Henderson et al, JACS (2009), 131: 4176-4177).
Regarding claim 1, Kahn teaches co-transfecting neurons with a plasmid for the expression of TagRFP-a-tubulin (i.e., a first polynucleotide encoding a first protein assembly monomer under control of a first promoter) and a plasmid for the expression of photoactivatable GFP (PA-GFP)-a-tubulin (i.e., a second polynucleotide encoding a second protein assembly monomer under control of a first promoter) (Fig 4; page 14, ¶4). Kahn teaches monitoring the dynamics of the microtubules after laser ablation of the microtubule (i.e., a cellular event) (Fig 4e). Kahn teaches the laser ablation leads to a visualizable change in the location of the PA-GFP-a-tubulin and TagRFP-a-tubulin (Fig 4e).
Regarding claim 2, Kahn teaches laser ablation of the microtubule (i.e., exposure to light) (Fig 4e).
Regarding claim 4, Kahn teaches the monomers are PA-GFP-a-tubulin and TagRFP-a-tubulin (Fig 4; page 14, ¶4). Kahn teaches microtubules comprising -a-tubulin spontaneously polymerize at the plus end (page 13, ¶1).
Regarding claim 5, Kahn teaches PA-GFP-a-tubulin and TagRFP are monomers of endogenous microtubules (Fig 4).
Regarding claim 8, Kahn teaches the plasmid encoding TagRFP-a-tubulin was constructing by subcloning the a-tubulin coding sequence in a plasmid called bactin, and referenced Hoogenraad (page 14, ¶4). Hoogenraad teaches that the bactin vector comprises the bactin promoter (i.e., an activity-independent constitutive promoter) for transgene expression (Supp Methods, ¶2). Therefore, Kahn’s method inherently comprised a first protein assembly monomer whose expression is driven by an activity-independent constitutive promoter.
Regrading claim 18, Kahn teaches illuminating the cells with 405 nm light (i.e., a cellular event) which leads to a visualizable change in the appearance of the microtubule and the PA-GFP α-tubulin is only visualizable after illumination with 405 nm light (Fig 4a). Kahn is silent on whether illumination with 405 nm light cases a post-translational modification. Henderson teaches illumination with UV light causes a decarboxylation of the Glu222 residue (i.e., a post-translational modification) (page 4176, ¶5). Therefore, Kahn’s method inherently comprised a visualizable change of the protein monomer that is caused by a post-translational modification.
Regarding claim 19, Kahn teaches α-tubulin fused to RFP and PA-GFP (i.e., visualizable tags) (Fig 4).
Regarding claim 36, in Kahn’s method, the plasmid for the expression of photoactivatable GFP (PA-GFP)-a-tubulin can also be considered the first polynucleotide encoding a first protein assembly monomer under control of a first promoter (Fig 4; page 14, ¶4). Kahn teaches PA-GFP fused to α-tubulin is activated by illumination with a 405 nm laser (i.e., is light-gated tag) (page 15, ¶9).
Regarding claim 41, the RFP and PA-GFP in Kahn’s method are genetically-encoded protein-based visualizable tags (page 14, ¶4), and are therefore inherently susceptible to cleavage by a protease
Regrading claim 47, Kahn teaches illuminating the cells with 405 nm light (i.e., a cellular event) which leads to a visualizable change in the appearance of the microtubule and the PA-GFP α-tubulin is only visualizable after illumination with 405 nm light (Fig 4a).
Regarding claim 49, Kahn is silent on whether illumination with 405 nm light causes a post-translational modification. Henderson teaches illumination with UV light causes a decarboxylation of the Glu222 residue (i.e., a post-translational modification) (page 4176, ¶5). Therefore, Kahn’s method inherently comprised a visualizable change of the protein monomer that is caused by a post-translational modification.
Regarding claim 100, options (i), (ii), and (iii), Kahn teaches co-transfecting neurons with a plasmid for the expression of photoactivatable GFP (PAGFP)-a-tubulin (i.e., a first polynucleotide encoding a first protein assembly monomer fused to a light-gated tag under control of a first promoter) and a plasmid for the expression of TagRFP-a-tubulin (i.e., a second polynucleotide encoding a second protein assembly monomer fused to a visualizable tag under control of a second promoter) (Fig 4; page 14, ¶4).
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.
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 14, 20, 25-26 and 34-35 are rejected under 35 U.S.C. 103 as being unpatentable over Kahn (Kahn et al., Nature Communications (2018), 9:2773, pages 1-17), as applied to claims 1-2, 4-5, 8, 18-19, 36, 41, 47, 49 and 100 above, and further in view of Singh (Singh et al., JACS (2013), 135: 6184-6191).
The teachings of Kahn are recited above as for claims 1-2, 4-5, 8, 18-19, 36, 41, 47, 49 and 100, and incorporated here.
Kahn does not teach monitoring multiple cells simultaneously. Kahn does not teach a-tubulin fused to a visualizable tag that binds to a fluorescent dye.
Singh teaches visualizing microtubules in cells by transfecting cells with a plasmid encoding a-tubulin fused to a HaloTag (i.e., a protein assembly monomer fused to a visualizable tag) and incubating the cells with fluorescent dyes (Fig 6). Singh teaches the HaloTag domain is a small protein that is readily expressed as a chimera with the protein of interest (page 6184, ¶1). Singh teaches the HaloTag binds fluorescent dyes, allowing visualization of the tagged protein (page 6184, ¶1). Singh teaches multiple cells expressing a-tubulin-HaloTag can be imaged simultaneously (Fig 7a). Singh teaches expressing two different proteins tagged with HaloTag, expressing the proteins at different times (i.e., different promoters), and incubating the cells with different fluorescent dyes at two times (i.e., t1 and t2) 48 hours apart (Fig 7).
Regarding claims 14 and 20, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted the RFP and/or PA-GFP tag fused to a-tubulin with the HaloTag taught in Singh. It would have amounted to the simple substitution of one known genetically-encoded tag used in live cell fluorescence microscopy to image protein dynamics in cells for another by known means to yield predictable results. The skilled artisan would have expected that that a plasmid encoding a-tubulin-HaloTag could be delivered to Kahn’s neurons along with another plasmid, because Singh demonstrates co-transfection of two different plasmids, one of which encodes a-tubulin-HaloTag. The skilled artisan would have also expected that multiple cells could be monitored at one time using the HaloTag technology because Singh demonstrates visualizing a wider field of cells expressing a-tubulin-HaloTag and labeled with the fluorescent dye. Because the prior art recognizes the equivalence of RFP, PA-GFP and HaloTags for the purpose of visualizing microtubules in cells, an express suggestion to substitute one equivalent component or process for another is not necessary to render such substitution obvious. MPEP 2144.06.II. Nevertheless, the skilled artisan would have been motivated to make the substitution because Singh teaches the HaloTag can be used to visualize proteins at different times by incubating with different fluorescent dyes.
Regarding claim 25-26 and 35, Singh teaches incubating the cells expressing a-tubulin-HaloTag with cyan ODS HaloTag ligand htS2SY (i.e., a first fluorescent dye that binds the visualizable tag for a period of time) (Fig 7; page 6186, ¶5). Singh teaches incubating the cells expressing a-tubulin-HaloTag a second time with cyan red HaloTag ligand htS2YKY (i.e., a second fluorescent dye that binds the visualizable tag for a period of time) (Fig 7; page 6186, ¶5). Singh teaches a-tubulin was labeled with the second fluorescent dye based on the overlap of fluorescence of the cyan and red dyes (i.e., determine the location of the a-tubulin monomers) (Fig 7).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have specifically substituted the PA-GFP tag fused to a-tubulin with the HaloTag taught in Singh. It would have amounted to the simple substitution of one known genetically-encoded tag used or live cell fluorescence microscopy to image protein dynamics in cells for another by known means to yield predictable results. The skilled artisan would have been motivated specifically substitute PA-GFP with the HaloTag because the HaloTag technology not only allows timed activation of fluorescence similar to the PA-GFP, but also allows multiple timed activation by using different fluorescent dyes as taught in Singh. Using multiple dyes would allow the skilled artisan to label all microtubules using constitutively expressed RFP-a-tubulin and sequentially label nascent a-tubulin with different dyes to study microtubule monomer turnover.
Regarding claim 34, Singh teaches HaloTag technology can be used with at least three different fluorescent dyes that fluoresce at different wavelengths (Fig 2).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have incubated the cells expressing RFP-a-tubulin and a-tubulin-HaloTag rendered obvious above with a third fluorescent dye to continue to sequentially label nascent a-tubulin with different dyes to study microtubule monomer turnover.
Allowable Subject Matter
Claim 59 is allowed. Claims 9-10, 22, 24 and 27-30 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter:
Claims 9-10, 22, 24 and 59 require driving expression of one of the protein assembly monomers using a promoter that is responsive to the cellular event being profiled. Claims 27-30 require the protein assembly to be stable enough such that the fluorescent dyes used to visualize the tags produce discernable colored bands in the assembly. Neither of these features are taught or suggested in the prior art for the following reasons.
Regarding claims 9-10, 22, 24 and 59, the closest prior art is Kahn in view of Singh, whose teachings are recited above in paragraphs 45-56, 62, 64 and 66. Briefly, Kahn teaches using two different fluorophores fused to tubulin monomers for visualizing microtubules dynamics in vivo. For reasons stated above in paragraphs 63 and 65, it would have been obvious to substitute the photoactivatable GFP tag with the HaloTag for viewing monomer turnover in microtubules. In the methods of both Kahn and Singh, expression of the tagged-tubulin monomers is constitutive. Although inducible promoters are well-known in the art, there is no suggestion in Kahn and Singh to induce expression of the tagged-tubulin monomers. Typically, inducible promoters are used when a tagged protein interferes with a cellular function, causing toxicity. See e.g., Goodson et al., Cold Spring Harb Protoc (2010); doi:10.1101/pdb.top85. However, there is no indication in Kahn or Singh that expression of RFP-, PA-GFP- or HaloTag-tagged tubulin interferes with microtubule dynamics. As such, it would have not have been obvious to include an inducible promoter in general, let alone a promoter that is activated by any cellular event that is meant to influence microtubule dynamics.
Regarding claims 27-30, although it is obvious to generally substitute the PA-GFP with the HaloTag fused to a-tubulin, which requires the addition of fluorescent dyes, it is highly unlikely that the use of different dyes would produce a colored band in the microtubules. Microtubules are dynamic structures that can polymerize and depolymerize over the course of a cell cycle. See e.g., Desai and Mitchison, Annu Rev. Cell Dev. Biol. (1997), 13: 83-117. Singh teaches waiting 48 hours between the addition of a first dye and a second dye. While use of the HaloTag would be useful for examining turnover and distribution of tubulin as it is incorporated into lengthening and then removed from shortening microtubules, the HaloTag would not be useful for examining bands in microtubules, since they aren’t stable enough in living cells to monitor through relatively longer cellular events like cell division.
Applicant demonstrates HaloTag-iPak4 monomers can form extremely stable filaments and can incorporate different dyes as the filaments grow to form bands in the filaments (Fig 2C). Li also uses iPak4 filaments and incorporates both GFP and iron-binding domain into the filaments. However, Li is directed to using the iPak4 crystals for magnetically manipulating the cells. There is no suggestion in Li or in the prior art to use two different fluorophores to label different portions of the iPak4 crystal.
Finally, the idea of recording cellular history is not a new one. Askary reviews previously developed methodologies to record cellular events/histories (Askary et al., Nature Reviews Genetics (2025), 26: 203-222). Importantly, Askary notes that before 2023, the vast majority of methodologies have used DNA to record the data given it’s relative stability in cells and only since 2023 have protein-based mechanisms been used (page 204, ¶5). A few attempts in the prior art that have used protein time-stamps were specifically for disease-related proteins, such as amyloid deposition, which do not appear to be applicable to monitoring generic cellular events (Condello et al., Scientific Reports (2011), 1:19, pages 1012). Linghu, whose teachings are recited above, also uses fluorescent biosensors as quasi-time stamps to record and measure cell signaling events, however the base proteins do not form filaments, and instead form amorphous aggregates or “puncta”. A thorough search of the prior art did not uncover using any known filament-forming protein such as microtubules to be used as a cell-recording device by forming distinct and stable bands that relate to the cellular events. As such it would not have been obvious to modify Li’s use of the iPak4 filaments to record information through time-differential protein labeling.
Conclusion
Claim 59 is allowed. Claims 9-10, 22, 24, 27-30 are objected to. Claims 1-2, 4-5, 7-8, 14, 18-20, 25-26, 34-36, 41, 47, 49, 53, and 100 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571)272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. 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.
/CATHERINE KONOPKA/Examiner, Art Unit 1635