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 .
Claims status
Claims 1-20 are pending following the Reply filed 04/10/2026. Claims 15-19 are withdrawn. Claims 1-14 and 20 are presently considered.
Election/Restrictions
Applicant’s election without traverse of Invention I, claims 1-14 and 20, in the reply filed on 04/10/2026 is acknowledged.
Claims 15-19 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 04/10/2026.
Priority
Applicant’s claim for benefit under 35 U.S.C. 119 (e) of Provisional application No. 63/425,294 filed on 11/14/2022 is acknowledged. The present application and all claims are being examined with the earliest effective filing date of 11/14/2022.
Information Disclosure Statement
The information disclosure statement (IDS) filed on 02/15/2024 has been considered by the examiner.
Claim Interpretation
During patent examination, the pending claims must be "given their broadest reasonable interpretation consistent with the specification." The Federal Circuit’s en banc decision in Phillips v. AWH Corp., 415 F.3d 1303, 1316, 75 USPQ2d 1321, 1329 (Fed. Cir. 2005) expressly recognized that the USPTO employs the "broadest reasonable interpretation" standard:
The Patent and Trademark Office ("PTO") determines the scope of claims in patent applications not solely on the basis of the claim language, but upon giving claims their broadest reasonable construction "in light of the specification as it would be interpreted by one of ordinary skill in the art." In re Am. Acad. of Sci. Tech. Ctr., 367 F.3d 1359, 1364[, 70 USPQ2d 1827, 1830] (Fed. Cir. 2004). Indeed, the rules of the PTO require that application claims must "conform to the invention as set forth in the remainder of the specification and the terms and phrases used in the claims must find clear support or antecedent basis in the description so that the meaning of the terms in the claims may be ascertainable by reference to the description." 37 CFR 1.75(d)(1).
Claim 3 recites the limitation, “wherein the first MinDE pair is orthogonal to the second MinDE pair”. In view of the instant specification (see pg. 19, para. [0086]):
As used herein an ‘orthogonal pair’ of MinDE refers to a circumstance where all of the following conditions are true: the first MinD and first MinE are reactive and exhibit the behaviors described herein; the second MinD and the second MinE are reactive and exhibit the behaviors described herein; the first MinD and the second MinE are not reactive and do not exhibit the behaviors described herein; and the second MinD and the first MinE are not reactive and do not exhibit the behaviors described herein.
Therefore, it is reasonably interpreted from applicant’s disclosure that when the first MinDE pair is “orthogonal” to the second MinDE pair, neither protein from the first pair is reactive with either protein of the second pair. It should also be noted that the “behaviors” of MinD and MinE proteins that are “reactive” to one another are well known in the art, as further discussed under 35 U.S.C. 102 and 35 U.S.C. 103 below.
Claim 9 recites the limitation, “wherein a functional moiety is coupled to the first MinD and/or the first MinE”. In view of the instant specification (see pgs. 26-27, para. [0120]):
A functional moiety comprises any such molecule which provides activity or purpose within the cell. By way of example, and not limitation, functional moieties may include mechanisms for, payload delivery, amplification of biochemical signals or frequency-modulation. Function moieties may further comprise transport, signaling, localization, cell growth, division or replication, metabolism or energy production or utilization molecules. Functional moieties may further comprise those which react to a report cost-cell signaling activities as well as those which self-organize host-cell activities to generate new cell behaviors. (Emphasis added)
Hence, the limitation of “a functional moiety” is not particularly limited, so long as it is a molecule known to have some activity. Therefore, the broadest reasonable interpretation of the claim is that the first MinD and/or the first MinE are coupled to any molecule having any function. Further, the term “coupled” is given its plain meaning, wherein the recited structures (MinD and/or MinE) must be physically fused or attached to the functional moiety.
Claim 10 recites the method step of, “adding an activating agent to the cell, thereby providing coupling between the functional moiety and a peripheral agent, thereby coupling the peripheral agent to the first MinD and/or the first MinE”. In view of the instant specification (see pg. 25, para. [0117]):
In some cases, the functional moiety is not the end-target for coupling to the spatial and temporal distribution within a cell and the actual end-target is a peripheral molecule that needs to be bound to the functional moiety by way of an activating agent. In these cases, the "signal" that is desired for coupling to the spatial and temporal distribution is turned "off" when the activating agent is absent and gets turned "on" when the activating agent is added. This is illustrated herein with rapamycin mediated induction of a PPI (Example 1). Other possibilities include, light (using an optogenetic interaction pair); for example a Lov domain and its associated light-dependent interaction peptide; or Phy/Pif, other chemically induced dimerizes (Auxin interaction-domain) or reverse-dimerizes (developed by Takara) or induction of a competitor to peel it off- that is, interactions can be held in place by a low-affinity leucine zipper and displaced by inducible expression (through a TetON system) of a higher affinity leucine zipper interaction.
It should be noted that the paragraph above contains the only explicit mention of the term “peripheral agent”. Therefore, the “peripheral agent” is not limited to any particular set of structures, and is interpreted to be any molecule that binds to the functional moiety when the activating agent is added to the eukaryotic cell. Further, in view of the description above, the activating agent is interpreted to be any molecule (e.g., rapamycin) or stimulus (e.g., light) that contributes to the interaction between the peripheral agent and the functional moiety.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(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.
Claim(s) 1, 4-5, 7, 9 and 20 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ramm, et al. (A diffusiophoretic mechanism for ATP-driven transport without motor proteins, Nat. Phys. 17, 850–858, 2021; cited on Form 892), hereafter, “Ramm”.
Regarding claim 1, Ramm teaches that the healthy growth and maintenance of a biological system depends on the precise spatial organization of molecules within the cell through the dissipation of energy. Ramm teaches that reaction–diffusion mechanisms can facilitate this organization, as can directional cargo transport orchestrated by motor proteins, by relying on specific protein interactions. However, transport of material through the cell can also be achieved by active processes based on non-specific, purely physical mechanisms, a phenomenon that remains poorly explored. Here, using a combined experimental and theoretical approach, Ramm discloses a hidden function of the Escherichia coli MinDE protein system: in addition to forming dynamic patterns, this system accomplishes the directional active transport of functionally unrelated cargo on membranes. See Abstract.
Ramm teaches that the Min system, a paradigmatic model for pattern formation in biology, regulates the site of cell division in E. coli, and the core of this reaction–diffusion system consists of only two proteins, the ATPase MinD and the ATPase activating protein MinE, which interact and reversibly bind to the membrane (pg. 850, col. 1, para. 2). In vitro, MinDE proteins form travelling surface waves or quasi-stationary patterns on planar artificial membranes and exhibit oscillations when geometrically confined, and these dynamics can provide spatial cues for particular proteins (see pg. 850, col. 1, para. 2). Exploiting these effects, Ramm revealed that MinDE can even spatially sort different cargo species (see pg. 850, col. 2, para. 2).
Ramm’s findings raised the question of whether MinDE-dependent transport via diffusiophoresis could occur in vivo, and thus, Ramm reconstituted MinDE oscillations together with inert model peripheral membrane proteins (mCh-MTS) in the evolutionary distant fission yeast, Schizosaccharomyces pombe (see pg. 855, col. 2, para. 3). Ramm found that in this in vivo model system, MinDE formed dynamic patterns with similar wavelength and velocity as in its native host E. coli and, spatiotemporally regulated mCh-MTS proteins on intracellular membranes (see pg. 856, col. 1, para. 1). Ramm discloses that a plasmid was employed for the co-expression of sfGFP-MinD and MinE (fluorescently-tagged MinD and MinE) in the yeast cell (see pg. 872, Extended Data Fig. 9).
Therefore, Ramm teaches the expressing of a MinDE pair comprising a first MinD and a first MinE in a Eukaryotic cell (yeast), which meets the claim.
Regarding claim 4, Ramm discloses that the MinD was bound to an sfGFP fluorescent reporter molecule (sfGFP-MinD), as discussed above. Ramm discloses imaging data showing the visualization of MinD dynamics within the cell using sfGFP, including the wavelength and velocity of travelling waves (see pg. 872, Extended Data Fig. 9). Therefore, Ramm teaches a reporter molecule coupled to the MinD, thereby providing at least partial visualization of the spatial and temporal distribution of molecules with the cell.
Regarding claim 5, Ramm discloses imaging data showing the visualization of MinD dynamics within the cell (see pg. 872, Extended Data Fig. 9), which were obtained via microscopy (see “Microscopy” on pg. 860, col. 1, para. 4). Therefore, Ramm teaches the further limitation of imaging the cell.
Regarding claim 7, Ramm teaches that MinDE proteins form travelling surface waves or quasi-stationary patterns and exhibit oscillations when geometrically confined (see pg. 850, col. 1, para. 2). Ramm discloses imaging of MinDE travelling waves and pole-to-pole like oscillations in a yeast cell (see pg. 872, Extended Data Fig. 9). Therefore, Ramm teaches the spatial and temporal distribution of molecules within the cell directed by the MinDE included travelling waves, which meets the claim.
Regarding claim 9, Ramm discloses that the MinD was attached to an sfGFP reporter molecule, as previously discussed. Ramm discloses imaging data showing the visualization of MinD dynamics within the cell, provided by the reporter molecule (see pg. 872, Extended Data Fig. 9).
As discussed under Claim Interpretation: The “functional moiety” is interpreted to be any molecule having any function that is attached to MinD and/or MinE.
Therefore, Ramm discloses the attachment of an sfGFP reporter to MinD, which meets the limitation of “wherein a functional moiety is coupled to the first MinD, thereby coupling the functional moiety to the spatial temporal distribution of molecules within the cell”.
Regarding claim 20, Ramm discloses expressing a MinDE pair comprising a first MinD and a first MinE in a Eukaryotic cell (yeast), as discussed regarding claim 1. As Ramm teaches the MinDE pair were derived from E. coli, they meet the limitation of a “non-natively expressed protein pair”.
Regarding the method step of “a) observing spatial and temporal distribution of molecules with a cell including… whole-cell oscillations [or] travelling waves”, Ramm discloses imaging data showing the visualization of MinD dynamics within the cell (as discussed regarding claim 4), wherein said imaging included MinDE travelling waves as well as pole-to-pole like oscillations in a yeast cell (as discussed regarding claim 7).
Regarding the limitation of a “functional moiety”, Ramm discloses expressing the pair of proteins within the cell (as discussed regarding claim 1), and the attachment of a sfGFP fluorescent molecule to MinD (as discussed regarding claim 9).
Therefore, Ramm teaches all the limitations of the claim.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ramm, as applied to claims 1, 4-5, 7, 9 and 20 above, and as further evidenced by Moebs, et al. (Traveling Waves. In University Physics Volume 1. Chapter 16.1. Houston, TX: OpenStax (2016); cited on Form 892), hereafter, “Moebs”.
Regarding claim 6, Ramm discloses that in the in vivo (yeast) model system, MinDE formed dynamic patterns with similar wavelength and velocity as in its native host E. coli and, spatiotemporally regulated mCh-MTS proteins on intracellular membranes, as discussed above. Ramm discloses analysis of “e”, the wavelength and “f”, wave velocity obtained from manually fitting the kymographs of cells displaying MinDE travelling waves (see pg. 872, Extended Data Fig. 9). Therefore, Ramm teaches that the MinDE generate traveling waves that were identified by their wavelength and velocity, which inherently comprise a frequency (i.e., v = f x λ, as disclosed by Moebs at pg. 5, para. 1). Hence, the imaging methods used by Ramm reasonably meet the limitation of being “frequency-specific”. As cells having the MinDE pairs would be expected to generate these waves, while others would not, it would have been obvious to have used these imaging techniques to identify which cells are expressing the MinDE pair. As the fluorescent imaging techniques disclosed by Ramm (see, e.g., pg. 856, Fig. 5; pg. 860, col. 1, para. 4, “Microscopy”; pg. 872, Extended Data Fig. 9) were known in the art, the results of identifying MinDE-expressing cells based on the frequency of their travelling waves was within the ordinary skill in the art at the time of filing. Therefore, a person of skill would have recognized that the results of identifying cells according to the frequency of travelling waves generated by the MinDE pair would have been predictable, and there would have been a reasonable expectation of success.
Claim(s) 2-3 and 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ramm (previously cited), as applied to claims 1, 4-5, 7, 9 and 20 above, and further in view of Ramm, et al. (The MinDE system is a generic spatial cue for membrane protein distribution in vitro. Nat Commun. 2018 Sep 26;9(1):3942; cited on Form 892), hereafter, “Ramm 2018”, Karnicolas, et al. (Designing orthogonal signaling pathways: how to fit in with the surroundings. Proc Natl Acad Sci USA. 2012 Apr 3;109(14):5140-1; cited on Form 892), hereafter, “Karnicolas” and Huidobro et al. (Synthetic spatial patterning in bacteria: advances based on novel diffusible signals. Microb Biotechnol. 2022 Jun;15(6):1685-1694; cited on Form 892), hereafter, “Huidobro”.
Regarding claim 2, Ramm teaches that the spatial organization of molecules within the cell can be facilitated by reaction–diffusion mechanisms (see Abstract). Ramm teaches that the Min system is a paradigmatic model for pattern formation in biology, and the core of this reaction–diffusion system consists of MinD and MinE, which interact and reversibly bind to the cell membrane (pg. 850, col. 1, para. 2). In vitro, MinDE proteins form travelling surface waves or quasi-stationary patterns and exhibit oscillations, and these dynamics can provide spatial cues for particular proteins (see pg. 850, col. 1, para. 2).
Ramm’s findings raised the question of whether MinDE-dependent transport via diffusiophoresis could occur in vivo, and thus, Ramm reconstituted MinDE oscillations together with inert model peripheral membrane proteins (mCh-MTS) in the evolutionary distant fission yeast, Schizosaccharomyces pombe (see pg. 855, col. 2, para. 3). Ramm found that in this in vivo model system, MinDE formed dynamic patterns with similar wavelength and velocity as in its native host E. coli and, spatiotemporally regulated mCh-MTS proteins on intracellular membranes (see pg. 856, col. 1, para. 1). Ramm discloses that a plasmid was employed for the co-expression of sfGFP-MinD and MinE (fluorescently-tagged MinD and MinE) in the yeast cell (see pg. 872, Extended Data Fig. 9). Hence, Ramm teaches the expressing of a MinDE pair comprising a first MinD and a first MinE in a Eukaryotic cell (yeast), as discussed regarding claim 1.
Ramm does not explicitly teach the method further comprising expressing a second MinDE pair comprising a second MinD and a second MinE in the eukaryotic cell.
However, Ramm also teaches that diffusiophoretic transport is presumably not a special feature of the E. coli MinDE system or reaction–diffusion systems in general, but can potentially be exerted by any active system producing concentration gradients (see pg. 857, col. 1, para. 1). For example, such a mechanism could be underlying the secretion-induced protein patterning that has been observed in fission yeast or be at play for the plethora of intracellular (actin) waves in eukaryotes (see pg. 857, col. 1, para. 1).
Ramm discloses another recent example of the Escherichia coli Min system in vitro, citing Ramm 2018 (see pg. 850, col. 1, para. 1; cite no. 7).
Ramm 2018 poses the question whether related reaction–diffusion systems, such as ParABS systems, Cdc42 and PAR proteins, are also capable of regulating a large set of proteins by similar nonspecific interactions as MinDE, as this may point to a generic mechanism of coupling large-scale molecular rearrangements and gradient formation to ATP consumption (see pg. 2, col. 2, para. 2). Using an in vitro reconstitution assay, Ramm 2018 showed that MinDE self-organization can spatially regulate a variety of functionally completely unrelated membrane proteins into patterns and gradients, and that the MinDE system can spatiotemporally control a much larger set of proteins than previously known (see Abstract). Ramm 2018 further designed a model peripheral membrane protein, mCh-MTS(BsD), consisting of the mCherry monomeric, fluorescent protein and a C-terminal amphipathic helix, the MTS from B. subtilis MinD (see pg. 2, col. 2, para. 2). Ramm teaches that this MTS is well-characterized and localizes other fluorescent proteins to the inner membrane in E. coli, but is unlikely to specifically interact with MinDE (see pg. 2, col. 2, para. 2). Ramm 2018 states that this work lays the foundation to apply this simplistic regulatory mechanism for positioning artificial division machineries and chromosomes in constructing a synthetic cell from the bottom up, and further speculates that other reaction-diffusion systems may be capable of regulating various proteins on their respective matrix (see pg. 12, col. 2, para. 2).
Therefore, Ramm 2018 suggests that reaction-diffusion systems that are analogous to the E. coli MinDE system may also be used to spatially regulate molecules within a cell in a similar manner. Ramm 2018 also suggests that the membrane-targeting sequence (MTS) from B. subtilis MinD localizes proteins to the membrane in E. coli without specifically interacting with E. coli MinDE.
Karanicolas teaches that the ability to rationally manipulate protein-protein interactions will represent a key enabling technology for understanding the properties of natural cellular networks, for building new biological responses into cells, and even as a starting point for therapeutic intervention in certain human diseases (see pg. 5140, col. 1, para. 1). Karanicolas teaches that successful integration into cells will require that engineered protein interactions (and later whole engineered pathways) can exist in parallel with endogenous cellular interactions (and pathways) without interference or crosstalk between the two: they must behave in a manner completely orthogonal to one another (see pg. 5140, col. 1, para. 1).
Karanicolas teaches that certain applications, such as introducing an enzyme/inhibitor cognate pair, may not require “wiring” the new interaction into the underlying cellular circuitry, because if a functionally similar protein pair exists in the host cell, extensive differences between the artificial pair may naturally lead to orthogonality (see pg. 5140, col. 1, para. 2). Karanicolas teaches that a new protein pair that is highly dissimilar to endogenous cognate pairs can be transferred from a separate organism or be produced by rational redesign (see pg. 5140, col. 1, para. 2). Karanicolas discusses a study in PNAS by Kapp et al., which reported the computational redesign of the GTPase/guanine nucleotide exchange factor (GEF) protein pair, Cdc42-Intersectin, in which Kapp demonstrated orthogonality between the wildtype GEF (Intersectin) and a GTPase (Cdc42) having a single pair of point-mutations (see pg. 5140, col. 1, para. 1; pg. 5140, col. 2, para. 2; pg. 5141, col. 3, para. 2). Karanicolas states:
[t]he demonstration that a single pair of carefully chosen point mutations is sufficient to confer orthogonality with little loss of function [by Kapp] leads one to wonder what range of orthogonal protein pairs could be created through similarly minimal changes in sequence. Accordingly, one can certainly envision extending the scale of this approach to build up complete orthogonal pathways that mimic and coexist alongside any number of endogenous cellular pathways in cells. (pg. 5141, col. 3, para. 3)
Therefore, Karanicolas suggests that the development of orthogonal protein pair interactions in cells can be useful for studying the properties of natural cellular networks, for building new biological responses into cells, and even as a starting point for therapeutic intervention in certain human diseases. Karanicolas also suggests that the orthogonality of such protein interactions may be achieved naturally, by selecting protein pairs that are dissimilar to one another. Finally, Karanicolas exemplifies an orthogonally functioning cdc42 protein, achieved by a single pair of point-mutations, which Ramm 2018 teaches to be part of a related reaction–diffusion system, capable of regulating proteins by similar nonspecific interactions as MinDE, as discussed above.
Huidobro teaches that advanced spatial control over gene expression may revolutionize fields such as medicine, through organoid or tissue engineering, and that, to date, foundational advances in spatial synthetic biology have often been made in prokaryotes, using artificial gene circuits (see Abstract). In Huidobro’s review, engineered patterns are classified into four levels of increasing complexity, ranging from spatial systems with no diffusible signals to systems with complex multi-diffusor interactions (see Abstract).
In addition to expanding our knowledge of developmental biology, Huidobro teaches that building a simple and programmable system is necessary for the synthesis of patterned tissues, organoids or biofilms for downstream biotechnology applications (see pg. 1685, col. 2, para. 3). To consider the problem of synthetic patterning systems systematically, Huidobro suggests grouping them into four levels according to their design characteristics: Level 0 circuits do not contain any synthetic signals that diffuse through normal Fickian diffusion, and spatial structure emerges by other processes, such as cellular growth; Level 1 systems rely on one or more diffusing components whose production is not dynamically regulated by the circuit; Level 2 systems incorporate a single diffusible component, which is dynamically regulated by the circuit components; and Level 3 systems use multiple dynamically regulated diffusible components (see pg. 1686, col. 1, para. 1).
Huidobro teaches that reaction–diffusion systems, where the diffusor is dynamically regulated by the circuit components (Level 2), have been engineered successfully, and have been observed to generate spatially synchronized oscillations and propagating waves (see pg. 1687, col. 1, para. 3). Level 3 systems are formed by reaction-diffusion circuits of at least two diffusors (see pg. 1687, col. 1, para. 4); however, multi-diffusor systems were historically held back by the lack of a diverse palette of well-characterized diffusible components (see pg. 1688, col. 1, para. 2). Huidobro teaches that the basic criteria that synthetic signaling molecules need to satisfy include orthogonality to other synthetic and endogenous cellular components, and it is also desirable that the signals are well-characterized and optimized for model-based rational engineering (see pg. 1688, col. 1, para. 4). Although this review focuses on E. coli, some studies indicate that these diffusors may be ported to other prokaryotes as well as some eukaryotes, including mammalian cells, for a wider range of applications (see pg. 1688, col. 2, para. 3). Huidobro teaches that while many interesting patterns have already been built, the potential for innovation is still great, particularly for multi-diffusor circuits, which could potentially show more diverse and complex spatiotemporal behaviors (see pg. 1690, col. 1, para. 2).
Taken together, a person of skill would have recognized that: (1) MinDE has been demonstrated to organize the spatial and temporal distribution of molecules when expressed in a eukaryotic cell; (2) the study of protein-protein interactions using orthogonal protein pairs may be useful for studying cellular properties and potential therapies for certain diseases in humans; (3) orthogonality may be achieved in reaction-diffusion systems using heterologous proteins or by rationally designed point-mutations; (4) the well-studied E. coli MinDE system is analogous to other reaction-diffusion systems used in other organisms; (5) MinD and MinE proteins from heterologous species, as demonstrated using the MTS of B. subtilis MinD, would be expected to be orthogonal to those derived from E. coli; and (6) multi-diffusor reaction-diffusion systems showing more complex spatiotemporal behaviors can be used as artificial circuits in eukaryotic cells, which may provide a wide range of biotechnology applications.
In view of Applicant’s Examples, cross-reactivity between MinD and MinE was investigated by transfecting equivalent amounts of plasmid DNA encoding fluorescently tagged versions of the proteins into HEK293T cells (see pg. 35, para. [0147]), and it was determined which combinations of MinDE pairs could provide two or more independent circuitries within the cell (see pg. 35, para. [0148]). There is no disclosure of any unexpected results, only that certain combinations of a first/second MinDE pair (from different organisms) were functional, while others were not (see pg. 17, para. [0077]).
Per MPEP 2144.04(VI)(B), the courts have held that “mere duplication of parts has no patentable significance unless a new and unexpected result is produced.” In re Harza, 274 F.2d 669, 124 USPQ 378 (CCPA 1960). In the instant case, two sets of MinDE pairs selected for their orthogonality to one another, or made to be orthogonal to one another, would be expected to function independently and to have the same function separately as they do in combination, and there is no evidence to suggest that the results of expressing more than one MinDE pair in a eukaryotic cell would have been unexpected. A person of skill would have expected that certain MinDE pairs, derived from different organisms, may interact (e.g., due to their close homology), while others may function independently (e.g., due to evolutionary divergence).
Therefore, it would have been obvious for a person of ordinary skill in the art to have expressed a second MinDE pair (i.e., reaction-diffusion system), because the prior art teaches that the design of orthogonal protein-protein interaction pathways is useful for the study of cellular functions and the discovery of therapeutics to treat human disease. One would have recognized that orthogonality in such multi-diffusor systems can be achieved by selecting a second MinDE pair that was heterologous to the first MinDE pair, e.g., from a separate organism, or by producing an orthogonal MinDE by rational redesign. One would have been particularly motivated to combine different MinDE pairs (i.e., reaction-diffusion systems), because Huidobro teaches that multi-diffusor circuits could potentially show more diverse and complex spatiotemporal behaviors for a wider range of applications, including in mammalian cells. Furthermore, Karanicolas teaches that the ability to manipulate such protein-protein interactions represents a key enabling technology for understanding the properties of natural cellular networks, for building new biological responses into cells, and as a starting point for therapeutic intervention in certain human diseases. A person of skill would have also recognized from Karanicolas that proteins having an analogous function to MinDE (i.e., Cdc42) have been successfully made to be orthogonal by a single pair of point mutations, and, from Ramm 2018, that other bacterial sources of MinDE proteins (i.e., B. subtilis MinD) have been shown to be naturally orthogonal to E. coli MinDE. As Ramm teaches the MinDE system to be a paradigmatic model for pattern formation in cells, and Ramm 2018 suggests that other reaction-diffusion systems may be capable of regulating various proteins in the same manner.
Therefore, a person of skill could have pursued the known potential solutions for providing a cell expressing multiple reaction-diffusion systems, and would have arrived at the solution of expressing a second MinDE pair with a reasonable expectation of success.
Regarding claim 3, it would have been obvious to have expressed a second MinDE pair that was orthogonal to the first MinDE pair for the reasons discussed regarding claim 2.
Regarding claim 8, Karanicolas teaches that the ability to rationally manipulate protein-protein interactions will represent a key enabling technology for understanding the properties of natural cellular networks, for building new biological responses into cells, and even as a starting point for therapeutic intervention in certain human diseases, as discussed above. Furthermore, Huidobro discloses that some studies indicate that diffusors may be ported to other prokaryotes as well as some eukaryotes, including mammalian cells, for a wider range of applications, as discussed above. Hence, it would have been obvious to have expressed the MinDE pair(s) in a mammalian cell.
Claim(s) 10-11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ramm, as applied to claims 1, 4-6, 7, 9 and 20 above, and further in view of Courtney, et al. (Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos. ACS Chem Biol. 2021 Nov 19;16(11):2434-2443; cited on Form 892), hereafter, “Courtney”.
Regarding claim 10, Ramm discloses that the MinD was bound to an sfGFP fluorescent reporter molecule (sfGFP-MinD), as discussed regarding claim 1. Ramm discloses imaging data showing the visualization of MinD dynamics within the cell using sfGFP (a green fluorescent protein), including the wavelength and velocity of travelling waves (see pg. 872, Extended Data Fig. 9). As discussed regarding claim 9, Ramm discloses the attachment of an sfGFP reporter to MinD, which meets the limitation of “wherein a functional moiety is coupled to the first MinD, thereby coupling the functional moiety to the spatial temporal distribution of molecules within the cell”.
Ramm does not explicitly teach the method further comprising “adding an activating agent to the cell, thereby providing coupling between the functional moiety and a peripheral agent”.
In view of the instant specification, “In some cases, the functional moiety is not the end-target for coupling to the spatial and temporal distribution within a cell and the actual end-target is a peripheral molecule that needs to be bound to the functional moiety by way of an activating agent” (see pg. 25, para. [0117]). The inventors used the chemical inducible heterodimerization system FKBP/FRB, wherein the FRB was fused to MinD and FKBP was fused to BFP (blue-fluorescent protein) (see pg. 31, para. 0137]). Therefore, it is interpreted that (1) the limitation of an activating agent includes rapamycin, (2) the limitation of a functional moiety includes FRB(-MinD), and (3) the limitation of a peripheral agent includes (FKBP-)BFP, such that when rapamycin (activating agent) is added to the cell, the dimerization of FKBP/FRB couples the BFP (a fluorescent reporter) with the activities of MinD within the cell.
Courtney teaches that rapamycin-induced dimerization of FKBP and FRB is the most
commonly utilized chemically induced protein dimerization system, and it has been extensively used to conditionally control protein localization, split-enzyme activity, and protein−protein interactions in general by simply fusing FKBP and FRB to proteins of interest (see Abstract). Courtney teaches that over the last several decades, chemical biologists have placed many biological processes under conditional control by employing small-molecule dimerizers of proteins (see pg. 2434, col. 1, para. 2). Courtney teaches that the most commonly utilized chemical inducer of dimerization (CID) is rapamycin, a natural product that binds to FK506
binding protein (FKBP) (see pg. 2434, col. 1, para. 2). Courtney teaches that this FKBP− rapamycin complex then binds to the FKBP−rapamycin binding domain of mTOR (FRB), forming a ternary complex (see pg. 2434, col. 1, para. 2). Courtney teaches that due to the small size of FKBP and FRB (12 and 11 kDa, respectively) and their strong binding affinity for complexation by rapamycin—essentially forming an irreversible interaction—these two domains have been extensively utilized as tools to conditionally control the protein−protein interaction of FKBP/FRB fusion proteins of interest (see pg. 2434, col. 1, para. 2). Moreover, the fast cellular diffusion and favorable pharmacokinetics in animals has led to a wide range of processes being placed under rapamycin-inducible control, including (i) regulation of protein translocation; (ii) reconstitution of split proteins or enzymes for activation (e.g., green fluorescent protein); and (iii) regulation of proteolytic stability (see pg. 2434, col. 1, para. 2).
Courtney teaches that numerous split-enzyme systems have been developed using
the FKBP/FRB dimerization pair, and the addition of rapamycin to these systems results in reconstitution of an active enzyme of interest (see pg. 2435, col. 2, para. 3). When these protein fusions were co-expressed in mammalian cells, minimal interaction between FKBP/FRB or the split-enzyme fragments occurs in the absence of the CID; however, following the addition of rapamycin, the ternary complex is formed, resulting in subsequent reconstitution of the active luciferase enzyme (see pg. 2435, col. 2, para. 3 to pg. 2436, col. 1, para. 1). Courtney also discloses that a nuclear translocation reporter was utilized to show that upon addition of rapamycin, the ternary complex formation of FKBP-rapamycin-FRB resulted in the co-localization of the fluorescent proteins as seen by the nuclear translocation of GFP (see pg. 2437, col. 2, para. 1).
Therefore, it would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by utilizing the FKBP-FRB dimerization system, as taught by Courtney, in order to provide conditional control of the MinDE pair within the eukaryotic cell. One would have been motivated to use the rapamycin-inducible FKBP/FRB proteins, because these domains have been extensively utilized as tools to control protein-protein interactions, and are also highly favored due to their small size, fast cellular diffusion, and favorable pharmacokinetics in animals. One would have recognized that the more direct reporter system taught by Ramm, wherein the MinD is coupled to a fluorescent reporter, could be modified to provide chemically-inducible control, i.e., by coupling the fluorescent reporter (e.g., GFP, BFP, sfGFP) and the MinD to FKBP and FRB respectively, enabling, for example, the fluorescent imaging of the system by the addition of rapamycin. As Courtney teaches this system has been extensively utilized for several decades in many similar processes involving protein-protein interactions, a person of skill would have recognized that the results of combining these teachings would have been predictable and there would have been a reasonable expectation for success. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 11, it would have been obvious to have used rapamycin as the activating agent for the same reasons discussed regarding claim 10.
Claim(s) 12-14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ramm, as applied to claims 1, 4-6, 7, 9 and 20 above, and further in view of GenBank CAD6010123.1 and GenBank ABC70501.1 (both cited on Form 892).
Regarding claim 12, Ramm teaches the expressing of an Escherichia coli MinDE pair comprising a first MinD and a first MinE in a Eukaryotic cell (yeast), as discussed regarding claim 1.
Ramm does not teach the amino acid sequence for Escherichia coli MinD or MinE.
However, Ramm teaches that the Min system is the paradigmatic model for pattern formation in biology and regulates the site of cell division in E. coli, and the core of this reaction–diffusion system consists of only two proteins, the ATPase MinD and the ATPase activating protein MinE, which interact and reversibly bind to the membrane (see pg. 850, col. 1, para. 2). Ramm teaches that the effects of MinDE can be exploited to spatially sort different cargo species (see pg. 850, col. 2, para. 2). Ramm found that in the in vivo yeast model system, MinDE formed dynamic patterns with similar wavelength and velocity as in its native host E. coli and spatiotemporally regulated mCh-MTS proteins on intracellular membranes (see pg. 856, col. 1, para. 1).
GenBank CAD6010123.1 is identified as minD derived from Escherichia coli (see Title and Organism). As shown in the following alignment, instant SEQ ID NO: 54 (top) is identical to the GenBank sequence (bottom):
PNG
media_image1.png
424
645
media_image1.png
Greyscale
GenBank ABC70501.1 is identified as minE derived from Escherichia coli (see Title and Organism). As shown in the following alignment, instant SEQ ID NO: 67 (top) is identical to the GenBank sequence (bottom):
PNG
media_image2.png
207
645
media_image2.png
Greyscale
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by selecting MinD and MinE proteins having the GenBank sequences above, because Ramm teaches the expression of a MinDE pair derived from E. coli in a yeast cell was sufficient to achieve the active transport of material through the cell using this paradigmatic system. One would have recognized that MinD and MinE proteins, particularly those from E. coli, were well known in the art and could be readily found in public databases. As the MinD and MinE amino acid sequences are identified as being sourced from E. coli, one would have recognized them to have been the same, or at least functionally equivalent, to the MinD and MinE proteins taught by Ramm. It should be noted that Ramm does not teach any modification to the wildtype sequences, and any wildtype E. coli MinDE pair would have been expected to have the same function. Therefore, one would have recognized that the results of using the MinDE protein pair according to GenBank would have been predictable, and there would have been a reasonable expectation of success. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 12, GenBank CAD6010123.1 is identical to instant SEQ ID NO: 54, as discussed above.
Regarding claim 13, GenBank ABC70501.1 is identical to instant SEQ ID NO: 67 as discussed above.
Conclusion
No claims are allowed.
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/DENNIS IGNATIUS ARMATO JR/Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651
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