DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of Group II (claims 2-6) in the reply filed on 01/21/2026 is acknowledged. The traversal is on the grounds that the inventions of Groups II (claims 2-6) and III (claims 7-9) share overlapping subject matter in that both require inactivating MUL1 as a core step. Applicant further asserts that inhibiting lipogenesis (Group II) is mechanistically related to protecting against obesity (Group III) since lipogenesis is a key pathway in obesity development. This is found persuasive, and the restriction requirement between Groups II and III is hereby withdrawn, and Groups II and III are hereby rejoined.
Because a claimed invention previously withdrawn from consideration under 37 CFR 1.142 has been rejoined, the restriction requirement between Groups II and III as set forth in the Office action mailed on 11/7/2025 is hereby withdrawn. In view of the withdrawal of the restriction requirement as to the rejoined inventions, applicant(s) are advised that if any claim presented in a divisional application is anticipated by, or includes all the limitations of, a claim that is allowable in the present application, such claim may be subject to provisional statutory and/or nonstatutory double patenting rejections over the claims of the instant application.
Once the restriction requirement is withdrawn, the provisions of 35 U.S.C. 121 are no longer applicable. See In re Ziegler, 443 F.2d 1211, 1215, 170 USPQ 129, 131-32 (CCPA 1971). See also MPEP § 804.01.
However, claim 1 stands withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 01/21/2026.
Accordingly, claims 2-9 are pending and under consideration.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63/348,687, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Application No. 63/348,687 fails to provide support for the claimed invention for the reasons given below in the rejections under 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph.
Therefore, claims 2-9 have an effective filing date of 06/02/2023, which is the filing date of the instant application.
Information Disclosure Statement
Receipt of information disclosure statements on 12/08/2025 and 01/16/2026 is acknowledged. The signed and initialed PTO-1449‘s have been mailed with this action.
The Examiner notes that numerous references are listed on pages 51-64 of the instant specification. The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The Examiner acknowledges the Petition to Accept Color Drawings under 37 CFR 1.84(a)(2), which was filed on 06/02/2023 and granted on 02/15/2024.
The replacement drawings (including color drawings) filed 09/20/2023 are objected to because:
Figure 16 illustrates the Promethion Cage, indicating that “Red text = Relevant to Behavior.” However, there is no red text shown in Figure 16. It would be remedial to clarify what text is relevant to behavior.
The drawing disclosed as “FIGS. 30A and 30B” is a single drawing with no separation of the indicated 30A and 30B. It would be remedial to clearly label what portion of the Figure is 30A and which is 30B, as disclosed in the instant specification (page 18, lines 28-33).
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (page 21, line 15; page 25, line 15; page 58, line 29). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The disclosure is further objected to because of the following informalities:
There is a typographical error at page 13, line 1. While the instant specification discloses “HE293 WT and MUL1(-/-) cells” therein, this appears to be a simple typographical error meant to disclose “HEK293” cells, as at page 12, line 28. It would be remedial to correct this typographical error.
Appropriate correction is required.
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant’s cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim Objections
Claims 2 and 5 are objected to because of the following informalities:
Claim 2 recites “a method for inhibiting lipogenesis in cells,” said method comprising several steps, including “applying a stain for identification of lipid droplets to the treated cells and untreated control cells,” followed by “identifying lipid droplets in the treated cells and in the untreated control cells using a stain capable of identification of lipids.” These steps overlap substantially despite differing slightly in wording. As is known to those of ordinary skill in the art, lipid droplets comprise lipids and can be visualized by applying neutral lipid dyes (or stains) such as BODIPY (reviewed in Mallela et al., 2019). Therefore, for purposes of presenting clear and concise claim language, it would be remedial to recite only one staining step for the identification of lipid droplets, which comprise lipids, as set forth above.
Claim 5 recites inactivation of “Mul1,” which while not technically improper is nonetheless inconsistent with the recitation of “MUL1” throughout the rest of the instant claim set. For purposes of internal consistency, it would be remedial to amend the instant claim to recite “MUL1” in place of “Mul1.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a) - Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 2-9 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 2-6 are drawn to a method of inhibiting lipogenesis in cells, said method comprising inactivating mitochondrial MUL1 E3 ubiquitin ligase (MUL1) in the cells. It is noted that this method is not limited to in vitro applications. Claims 7-9 are drawn to a method for protecting a subject against obesity induced by a high fat diet (HFD) by inactivation of mitochondrial MUL1 E3 ubiquitin ligase (MUL1). The rejected claims thus require inactivation of MUL1, wherein the agent inactivating MUL1 is not specifically defined. Thus, the claims necessarily encompass inactivators that are defined solely by their ability to function to inactivate MUL1, thereby inhibiting lipogenesis or protecting a subject against obesity induced by a high fat diet.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof. The specification describes inactivation of MUL1 with CRISPR-Cas9 in vitro (Figures 30A-B; page 21, lines 12-28; page 27, lines 3-18; page 29, line 28-page 30, line 15), as recited at instant claim 5. The mice carrying a whole-body inactivation of the MUL1 gene described in the instant specification are disclosed in Goyon et al., 2022 per the instant specification (page 37, line 18). Per Goyon et al., 2022, the MUL1-/- mice were produced via Cre/Lox recombination (lines 439-157). No description is provided of in vivo use of CRISPR-Cas9 to produce Mul1-/- mice, nor is any inactivator (or inhibitor) of MUL1 described other than the aforementioned in vitro application of CRISPR-Cas9.
Even if one accepts that the examples described in the specification meet the claim limitations of the rejected claims with regard to structure and function, the examples are only representative of in vitro inactivation of MUL1 with CRISPR-Cas9 and in vivo inactivation of MUL1 in mice via Cre/Lox recombination.
The results are not necessarily predictive of the broadly and functionally claimed inactivation of MUL1, including inactivation of MUL1 by administration of an inhibitor of MUL1, as recited at instant claim 9.
Thus, it is impossible for one to extrapolate from the few examples described herein those MUL1-inactivating agents that would necessarily meet the structural/functional characteristics of the rejected claims, particularly inhibitors of MUL1, as recited at instant claim 9. While the instant specification contemplates the development of chemical molecule inhibitors or therapeutic siRNA targeting MUL1 to be used against obesity and the accompanying metabolic disease (page 49, lines 8-10), such chemical molecule inhibitors are not disclosed in the instant specification, nor are they known in the prior art, as set forth below.
The prior art does not appear to offset the deficiencies of the instant specification in that it does not describe a set of MUL1-inactivating agents that would necessarily meet the structural/functional characteristics of the rejected claims. While Goyon et al., 2022 discloses whole-body inactivation of MUL1 in mice using Cre/Lox recombination (as set forth above) and Zhao et al., 2020 discloses knockdown of MUL1 expression with Mul1-targeted siRNA (page 1166, column 1, paragraph 2), these techniques are built on fundamentally different biological principles and cannot (on their own) be considered to describe a broad genus of MUL1-inactivating agents that necessarily meet the structural/functional characteristics of the rejected claims. In fact, the prior art appears to be entirely silent as to specific inhibitors of MUL1 (such as the chemical molecule inhibitors contemplated in the instant specification and set forth above), as recited at instant claim 9.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 2-9.
Claim Rejections - 35 USC § 112(a) - Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 2-9 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for inhibition of lipogenesis via CRISPR-Cas9-mediated inactivation of MUL1 in vitro, as well as inhibition of lipogenesis and protection against obesity induced by a high fat diet via Cre/Lox recombination-mediated inactivation of MUL1 in mice in vivo, does not reasonably provide enablement for inhibition of MUL1 via any other method, nor does it reasonably provide enablement for in vivo inactivation of MUL1 in human subjects in order to inhibit lipogenesis or protect against obesity induced by a high fat diet, as is encompassed by the instant claim set. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
It is noted that the instant disclosure does not provide literal or inherent support for the prior art methods addressed herein.
Enablement is considered in view of the Wands factors (MPEP 2164.01(A)). These include: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and the quantity of experimentation needed to make or use the invention. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention: Claims 2-6 are drawn to a method of inhibiting lipogenesis in cells, said method comprising inactivating mitochondrial MUL1 E3 ubiquitin ligase (MUL1) in the cells. Claims 7-9 are drawn to a method for protecting a subject against obesity induced by a high fat diet (HFD) by inactivation of mitochondrial MUL1 E3 ubiquitin ligase (MUL1). Dependent claim 9 specifically requires inactivation of MUL1 via administration of an inhibitor of MUL1, which encompasses chemical molecule inhibitors per the instant specification (page 49, lines 8-10). The nature of the invention is complex in that one must be able to make MUL1 inhibitors of undefined structure and/or inhibit MUL1 via any method in order to inhibit lipogenesis and/or protect a subject against obesity induced by a high fat diet.
Breadth of the claims: The claims broadly encompass the administration of compositions defined solely or primarily by function, wherein the compositions must be effective to inhibit MUL1 to inhibit lipogenesis and/or protect a subject against obesity induced by a high fat diet. Claims 2-6 recite administration of such inactivating agents/compositions to human or animal cells but do not limit this administration to in vitro applications. Claims 7-9 recite administration of said compositions to a subject and therefore are drawn to in vivo applications. The complex nature of the subject matter of this invention is greatly exacerbated by the breadth of the claims.
Guidance of the specification and existence of working examples: While the specification envisions the development of chemical molecule inhibitors or therapeutic siRNA for targeting MUL1 to treat obesity and the accompanying metabolic disease (page 49, lines 8-10), it does not teach any such chemical molecule inhibitors or therapeutic siRNAs. The specification does disclose in vitro CRISPR/Cas9-mediated knockout of MUL1 in HeLa cells (page 21, lines 12-28). Additionally, while the specification discloses whole-body MUL1(-/-) mice, the production of these mice is not disclosed in the instant specification. Rather, these mice are disclosed to have been produced in the study of Goyon et al., 2022 (page 37, line 18 of the instant specification) via Cre/Lox recombination (lines 439-157 of Goyon et al., 2022). The instant specification asserts that the in vitro and in vivo data disclosed therein establishes a new function of mitochondrial MUL1 E3-ubiquitin ligase in the regulation of lipogenesis and fatty acid oxidation, suggesting that MUL1 can be a very promising target for the development of chemical molecule, pharmacological inhibitors or therapeutic siRNA that could be used against obesity and the accompanying metabolic disease, such as nonalcoholic fatty liver disease, type 2 diabetes, heart or kidney disease, stroke, and certain kinds of cancers (page 49, lines 8-10; page 50, lines 16-19). However, no such chemical/pharmacological inhibitors or siRNAs are disclosed in the instant specification.
In fact, other than contemplating pharmacological targeting of MUL1 to treat obesity and accompanying metabolic disease, the specification offers no other guidance for the development and/or application of such inhibitors. As clearly stated in Genentech Inc. v. Novo Nordisk A/S (CAFC) 42 USPQ2d 1001: “Patent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable. See Brenner v. Manson, 383 U.S. 519, 536, 148 USPQ 689, 696 (1966) (stating, in the context of the utility requirement, that “a patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.”) Tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.” Applicant cannot rely on the knowledge of one skilled in the art to supply information on the novel aspects of the claimed invention, to which the instantly filed disclosure is largely silent as set forth above.
Predictability and state of the art: While the instant specification discloses in vitro CRISPR/Cas9-mediated knockout of MUL1 in human cells, it is silent as to in vivo CRISPR/Cas9-mediated knockout of MUL1, including in human subjects, as is encompassed by the instant claim set. As is known to those of ordinary skill in the art, applying CRISPR editing to patients in vivo is an underdeveloped and unpredictable topic in the current state of the art. As reviewed in Behr et al., 2021 and Liu et al., 2021, there are numerous hurdles to overcome before in vivo CRISPR therapy is commonplace. These hurdles include, but are not limited to, editing efficiency (discussed in section “Editing efficiency” of Liu et al., 2021), effective delivery of the editing machinery (discussed in section 6 of Behr et al., 2021 and section “Delivering methods” of Liu et al., 2021), off-target effects (discussed in section “Off-target effects” of Liu et al., 2021), and immunogenicity (discussed in section “Immunogenicity” of Liu et al., 2021). While in vivo CRISPR/Cas9-mediated editing in the liver has been documented, further investigation is needed as to off-target indels and long-term follow-up to establish long-term efficacy and toxicity (section 5.1 of Behr et al., 2021). The instant application is silent as to any proposals to overcome any of the hurdles set forth above, which would be necessary to apply in vivo CRISPR/Cas9 editing to human subjects to inhibit/inactivate MUL1 in order to inhibit lipogenesis and protect against obesity induced by a high fat diet.
Additionally, while the MUL1 knockout mice disclosed in the instant specification were produced via Cre/Lox recombination (Goyon et al., 2022), Cre/Lox recombination has not been documented in human subjects for in vivo gene knockout. With further regard to the knockout mice of the instant disclosure, the only data presented to support in vivo therapeutic benefit of inactivation/inhibition of MUL1 to inhibit lipogenesis and protect against obesity induced by a high fat diet were obtained using this mouse model. However, mice are an imperfect model for human diseases, particularly in the liver, which oversees certain essential pathways such as lipid metabolic processes (Jiang et al., 2020: page 8, paragraph 1). As disclosed in Jiang et al., 2020, humans and mice have substantially divergent gene regulation and thus, there are a significant number of biological processes that are subject to human-specific regulation and need to be carefully considered in the process of mouse to human translation (abstract). Thus, absent a showing to the contrary, one of ordinary skill in the art would not necessarily reasonably predict that in vivo mouse model findings related to lipid metabolic processes directly translate to the same in humans.
Furthermore, as set forth above, the prior art is entirely silent as to chemical molecule/pharmacological inhibitors of MUL1, as is envisioned and embraced by the instant application.
Amount of experimentation necessary: The quantity of experimentation required to carry out the full scope of the claimed methods is large. One could not solely rely upon guidance provided in the instant disclosure or in the prior art. As set forth above, the prior art is entirely silent as to chemical molecule/pharmacological inhibitors of MUL1, as is envisioned and embraced by the instant application. Thus, one would first be required to carry out screening assays to identify compounds capable of inhibiting MUL1. Given that such inhibitory or inactivating agents are broadly and functionally claimed (as set forth above), one would reasonably expect screening many different types of compounds that could be capable of inhibiting MUL1, including (but not limited to), siRNAs, shRNAs, antibodies, peptides, and chemical compounds. Next, one would be required to test each of the compounds for the ability to inhibit/inactivate MUL1 at a level sufficient to induce as least some therapeutic effect in a patient diagnosed with obesity. The success of one compound would not necessarily provide a reasonable expectation of success with another compound. For example, while siRNAs and shRNAs are both short, RNA-based agents for silencing gene expression, they are not completely interchangeable, as shRNAs have been reported to be significantly more potent than siRNAs at mediating knockdown when compared on a mole basis (McAnuff et al., 2007: abstract). Thus, optimizing the inactivating agent(s) targeting MUL1 for therapeutic applications as recited in the instant claim set would require substantial experimental effort to design, test, and optimize a staggering breadth of species of inactivating agents, with no guidance from the instant specification. Additionally, as set forth above, in vivo CRISPR therapy in human subjects is an underdeveloped method that still requires considerations regarding editing efficiency, effective delivery of the editing machinery, off-target effects, and immunogenicity, all to which the instant application is entirely silent.
In view of the breadth of the claims and the lack of guidance provided by the specification as well as the unpredictability of the art, the skilled artisan would have required an undue amount of experimentation to make and/or use the claimed invention. Therefore, claims 2-9 are not considered to be fully enabled by the instant disclosure.
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 2-6 are rejected under 35 U.S.C. 103 as being unpatentable over Calle et al., 2022 (hereinafter Calle; first published 30 May 2022) in view of Ameer et al., 2014 (hereinafter Ameer), Kim et al., 2018 (hereinafter Kim), Lee et al., 2012 (hereinafter Lee), and Eynaudi et al., 2021 (hereinafter Eynaudi).
With regard to claim 2, which recites “a method for inhibiting lipogenesis in cells comprising:
providing cells;
inactivating mitochondrial MUL1 E3 ubiquitin ligase (MUL1) in the cells;
treating a portion of the cells with a simulator of a high fat diet for a pre-determined period of time;
using a remaining portion of the cells as untreated control cells;
applying a stain for identification of lipid droplets to the treated cells and untreated control cells;
identifying lipid droplets in the treated cells and in the untreated control cells using a stain capable of identification of lipids;
visualizing lipid droplets identified in the treated cells and in the untreated control cells using an optical instrument; and
comparing an amount of stained lipid droplets visualized in the treated cells to an amount of stained lipid droplets visualized in the untreated control cells, wherein a decrease of stained lipid droplets in the treated cells indicates inhibition of lipogenesis in the treated cells,” Calle discloses that MUL1 is a mitochondrial protein with established roles in metabolic diseases (section 4.4). Additionally, expression of MUL1 is known to be upregulated in diet-induced obese mice displaying insulin resistance (Table 2). In fact, this induced upregulation of MUL1 is implicated in inducing insulin resistance in obese mice (Figure 2d; page 862, column 2, paragraph 2). These phenomena are all connected in that obesity is a metabolic disorder that is known to be associated with insulin resistance and aberrant de novo lipogenesis, as reviewed in Ameer (abstract; Figure 2; Sections 3, 5, and 6). Thus, Calle establishes that higher expression of MUL1 is associated with metabolic disorders such as obesity, which is also associated with aberrant de novo lipogenesis per Ameer. Therefore, prior to the effective filing date of the instant application, one of ordinary skill in the art would be aware that higher expression of MUL1 is associated with obesity and aberrant de novo lipogenesis based on the disclosures of Calle and Ameer and would thus reasonably predict that inactivating MUL1 would have the effect of reducing de novo lipogenesis and protecting against obesity. Methods of inactivating MUL1 Are known in the art. For example, Kim discloses inactivation of MUL1 by knocking out MUL1 in a cell line using the CRISPR/Cas9 system (abstract).
Methods for evaluating this prediction by assaying lipogenesis in vitro are well-known in the art. Lee discloses methods of assaying de novo lipogenesis in vitro in Huh-7 liver cells (abstract), wherein said Huh-7 cells are treated with experimental agents or vehicle controls (page 103, column 2, paragraph 2) and subsequently stained with BODIPY to visualize lipid droplets using microscopic imaging (page 103, column 2, paragraph 3; Figure 2), as instantly claimed. The vehicle control treatment disclosed in Lee is considered to read on the instantly claimed untreated control cells, as supported by Figure 2 of Lee. Lee further discloses that production of cytosolic lipid droplets is a major indicator of de novo lipogenesis and thus, an increase in cytosolic lipid droplet staining is associated with an increase in de novo lipogenesis (page 104, column 2, paragraph 2). One of ordinary skill in the art would therefore reasonably conclude that conversely, a reduction in cytosolic lipid droplet staining is associated with a decrease in de novo lipogenesis.
While Lee discloses treatment of Huh-7 liver cells with experimental agents or vehicle controls, these experimental agents are not the instantly claimed simulator of a high fat diet, which is associated with obesity per Eynaudi (page 9, column 1, paragraph 2). Eynaudi further discloses that oleic acid is an abundant component of both the Western diet and of high-fat diets and is associated with increased lipid droplet formation in HepG2 liver cells treated with oleic acid for 24 hours (page 2, column 1, paragraph 2; page 2, column 2, paragraph 2; page 9, column 1, paragraph 2; Figure 1). As set forth above regarding the disclosure of Lee, an increase in cytosolic lipid droplet staining (such as reported in Eynaudi following treatment of HepG2 liver cells with oleic acid) is associated with an increase in de novo lipogenesis. Therefore, the increased lipid droplet formation disclosed in Eynaudi is considered to reflect an increase in lipogenesis, as is also associated with high fat diet-induced obesity, as set forth above. Thus, it is considered that Lee and Eynaudi collectively disclose the methods of instant claim 2 with the exception of inactivation of MUL1, which is motivated by Calle, Ameer, and Zhao, as set forth above.
Accordingly, it is considered that Calle, Ameer, Kim, Lee, and Eynaudi collectively disclose the method of instant claim 2.
With regard to claim 3, which recites “providing cells [of the method of claim 2] includes providing at least one of human cells and animal cells,” as set forth above, Lee discloses in vitro assaying of lipogenesis in Huh-7, a human hepatoma cell line that maintains key features of hepatic lipid metabolism (page 103, column 1, paragraph 3). Thus, Lee discloses each and every additional limitation of instant claim 3.
With regard to claim 4, which recites “providing cells [of the method of claim 2] includes providing at least one of human liver cells,” as set forth above, Lee discloses in vitro assaying of lipogenesis in Huh-7, a human hepatoma cell line that maintains key features of hepatic lipid metabolism (page 103, column 1, paragraph 3). Thus, Lee discloses each and every additional limitation of instant claim 4.
With regard to claim 5, which recites “inactivating Mul1 includes inactivating Mul1 using CRISPR-Cas9,” as set forth above, Kim discloses in vitro inactivation of MUL1 by knocking out MUL1 in a cell line using the CRISPR/Cas9 system (abstract). Thus, Kim discloses each and every additional limitation of instant claim 5.
With regard to claim 6, which recites “treating a portion of the cells [of the method of claim 2] with a simulator of a high fat diet for a pre-determined period of time includes treating a portion of the cells with oleic acid for 24 hours,” as set forth above, Eynaudi discloses that oleic acid is an abundant component of both the Western diet and of high-fat diets and is associated with increased lipid droplet formation in HepG2 liver cells treated with oleic acid for 24 hours (page 2, column 1, paragraph 2; page 2, column 2, paragraph 2; page 9, column 1, paragraph 2; Figure 1). Thus, Eynaudi discloses each and every additional limitation of instant claim 6.
Given that Calle discloses that expression of MUL1 is known to be upregulated in high fat diet-induced obese mice displaying insulin resistance; that Ameer discloses that metabolic disorders such as obesity are also associated with aberrant de novo lipogenesis; that Kim discloses in vitro inactivation of MUL1 by knocking out MUL1 in a cell line using the CRISPR/Cas9 system; that Lee discloses methods of assaying de novo lipogenesis in vitro in Huh-7 liver cells; and that Eynaudi discloses that treatment of HepG2 liver cells with oleic acid (part of both the Western diet and of high fat diets and associated with obesity) for 24 hours is associated with increased lipid droplet formation, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to inactivate MUL1 in liver cells in vitro using methods such as CRISPR-Cas9 and provide said cells with a simulator of a high fat diet to predictably inhibit lipogenesis, as determined by subsequently assaying lipogenesis in said cells by monitoring lipid droplet formation, wherein decreased lipid droplet formation is associated with decreased lipogenesis. One would have been motivated to make such a modification in order to receive the expected benefit of inhibiting lipogenesis in cells provided with a high fat diet.
Claims 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Calle et al., 2022 (hereinafter Calle; first published 30 May 2022) in view of Goyon et al., 2022 (hereinafter Goyon).
With regard to claim 7, which recites “a method for protecting a subject against obesity induced by a high fat diet (HFD) by inactivation of mitochondrial MUL1 E3 ubiquitin ligase (MUL1), the method comprising:
providing an inactivator of MUL1; and
administering the inactivator of MUL1 to the subject, thereby inactivating MUL1 and protecting the subject against obesity induced by a high fat diet (HFD),” as set forth above, Calle discloses that MUL1 is a mitochondrial protein with established roles in metabolic diseases (section 4.4). Additionally, expression of MUL1 is known to be upregulated in diet-induced obese mice displaying insulin resistance (Table 2). In fact, this induced upregulation of MUL1 is implicated in inducing insulin resistance in diet-induced obese mice (Figure 2d; page 862, column 2, paragraph 2), as recited in the instant claim. Thus, Calle establishes that higher expression of MUL1 is associated with metabolic disorders such as obesity, particularly in animals wherein the obesity was induced by a high fat diet. Therefore, prior to the effective filing date of the instant application, one of ordinary skill in the art would be aware that higher expression of MUL1 is associated with obesity in animals fed a high-fat diet based on the disclosure of Calle and would thus reasonably predict that inactivating MUL1 would have the effect of protecting against obesity induced by a high fat diet.
Furthermore, methods of inactivating MUL1 are known in the art. For example, Goyon, discloses whole-body in vivo inactivation of MUL1 in mice using the Cre/Lox recombination system (lines 439-157).
Accordingly, it is considered that Calle and Goyon collectively disclose the method of instant claim 7.
With regard to claim 8, which recites “the subject [of the method of claim 7] is at least one of a human and an animal,” as set forth above, Calle discloses that expression of MUL1 is known to be upregulated in diet-induced obese mice displaying insulin resistance (Table 2), while Goyon discloses whole-body in vivo inactivation of MUL1 in mice using the Cre/Lox recombination system (lines 439-157). Given that mice are animals, it is considered that Goyon discloses each and every additional limitation of instant claim 8.
With regard to claim 9, which recites “the inactivator of MUL1 [of the method of claim 7] is an inhibitor of MUL1,” the Examiner notes that the term “inhibitor” is not clearly and specifically defined in the instant specification. Accordingly, under broadest reasonable interpretation, any agent that inhibits or inactivates MUL1 is considered to read on the instantly claimed inhibitor of MUL1. Thus, Cre/Lox recombination-mediated whole-body inactivation of MUL1 in mice (disclosed in Goyon and set forth above) is considered to read on the instantly claimed inhibitor of MUL1. Thus, it is considered that Goyon discloses each and every additional limitation of instant claim 9.
Given that Calle discloses that expression of MUL1 is known to be upregulated in high fat diet-induced obese mice, and that Goyon discloses in vivo inactivation of MUL1 in mice using the Cre/Lox recombination system, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to inactivate MUL1 in obese mice induced with a high fat diet to predictably protect against obesity induced by said high fat diet. One would have been motivated to make such a modification in order to receive the expected benefit of protecting mice against obesity induced by a high fat diet.
Conclusion
No claims are allowed.
Claims 2 and 5 are objected to.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637