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 .
1. The Amendment filed July 28, 2026 in response to the Office Action of July 28, 2026, is acknowledged and has been entered. Claims 1-19 are pending. Claims 3, 5 and 19 are amended. Claims 2, 8-18 remain withdrawn. Claims 1, 3-7 and 19 are currently being examined as drawn to the species of:
A. siRNA RNF167 inhibitor (claims 5 and 19);
B. Cancer/breast cancer (claims 3 and 4);
C. Method further requiring administering an anticancer agent that is a protein kinase B (AKT) inhibitor (claims 6 and 7).
New Rejections
(necessitated by amendments)
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
2. Claims 5 and 19 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. Claims 5 and 19 recite a GenBank Accession number as the sole means of identifying the claimed RNF167 nucleic acid sequence. The use of a GenBank accession number only to identify a particular molecule renders the claims indefinite because sequences of GenBank numbers are known to change and be revised over time. For example, see the attached lengthy revision history for GenBank Accession No. NM_015528.3, printed October 2026. The GenBank sequence is subject to revision.
Suggestion: Amending the claims to specifically and uniquely identify the RNF167 nucleic acid by SEQ ID NO can obviate the rejection.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
3. Claim 19 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 19 depends from claim 5. Claim 5 is limited to siRNA that “is at least about 95% homologous to a portion of the RNF167 nucleic acid sequence set forth in GenBank Accession No. NM_015528.3”. Claim 19 recites that the siRNA “is homologous to a portion of the RNF167 nucleic acid sequence set forth in GenBank Accession No. NM_015528.3”. The scope of siRNA in claim 19 is broader than in claim 5. Claim 19 encompasses siRNA having any percentage homology to a portion of the sequence set forth in GenBank Accession No. NM_015528.3, whereas claim 5 recites at least about 95% homology. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Maintained Rejection
(arguments added to address amendments)
Claim Rejections - 35 USC § 112
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.
4. Claims 1, 3-7 and 19 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The court in Wands states: "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue,' not 'experimentation.' " (Wands, 8 USPQ2d 1404). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighing many factual considerations." (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
The claims are drawn to a method of treating a disease in a subject, comprising administering a therapeutically effective amount of a ring finger protein 167 (RNF167) inhibitor to the subject;
wherein the disease is cancer that expresses CASTOR1 phosphorylated serine 14 and RNF167 and is breast cancer;
wherein the inhibitor is siRNA; and wherein the siRNA is at least about 95% homologous to a portion of a RNF167 nucleic acid sequence, wherein the RNF167 nucleic acid sequence is set forth in GenBank Accession No. NM_015528.3.
It is noted that amended claim 3 recites that the disease treated is cancer that expresses CASTOR1 phosphorylated at serine 14 and RNF167. The claim encompasses cancers that express CASTOR1 phosphorylated at S14 and express RNF167 at any level in any amount.
The specification discloses:
Example 1: RNF167 Activates mTORC1 and Promotes Tumorigenesis by Targeting CASTOR1 for Ubiquitination and Degradation
[0176] This Example shows that a low expression level of CASTOR1 is correlated with poor patient survival in numerous types of cancer including breast cancer, and that CASTOR1 is a substrate of RNF167. Furthermore, AKT-mediated phosphorylation of CASTOR1 facilitates its interaction with RNF167, leading to CASTOR1 ubiquitination and proteasome-dependent degradation. Additionally, CASTOR1 phosphorylation at S14 by AKT decreases its binding affinity to the GATOR2 complex. The phosphorylation and degradation of CASTOR1 collectively release the GATOR2 complex, activate mTORC1, and promote breast cancer progression. These findings reveal a novel mechanism by which cancer cells overcome the suppressive effect of CASTOR1 in the nutrient-deficient tumor microenvironment, and hence identify a potential novel therapeutic target for treating mTORC1-associated diseases including cancer.
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[0194] RNF167 mediates K29-linked polvubiquitination and degradation of CASTOR1 in response to growth factors: To reveal the environmental cue that activates mTORC1 by modulating the expression of CASTOR1, cells were deprived of either fetal bovine serum (FBS) or arginine. The kinetic analysis demonstrated that CASTOR1 protein level but not mRNA level was increased following 16 h of FBS deprivation in 293T cells, which was correlated with a decreased mTORC1 activity as shown by the reduced phosphorylation level of its downstream targets S6K and 4EBP1 (FIG. 1A, and FIG. 6A). As expected, the level of AKT activation was significantly reduced, which was noticeable at as early as 2 hrs but more obvious after 16 hrs following FBS deprivation (FIG. 1A). Hence, the level of CASTOR1 protein inversely trailed that of AKT activation following FBS deprivation.
[0195] The above results showed a negative correlation of AKT activation with the CASTOR1 protein level, which was strongly regulated by FBS deprivation but only marginally by arginine deprivation, suggesting an important regulatory role of growth factors in CASTOR1 protein level. Treatment with AKT inhibitor MK2206 in 293T cells upregulated CASTOR1 protein but not mRNA level, and decreased mTORC1 activation, mimicking FBS deprivation (FIG. 1C, and FIG. 6K). Because mTORC1 could be responsive to other nutrients such as leucine present the medium, the effect of leucine deprivation on CASTOR1 protein was further examined. Similar to arginine deprivation, chronic leucine deprivation activated AKT, reduced CASTOR1 mRNA and protein levels, and mTORC1 activity (FIGS. 6 k and 6L). Interestingly, a S6K1 inhibitor that decreased the pS6K but not p4EBP1 level, failed to activate AKT and reduce CASTOR1 protein level (FIG. 6L). Together these results suggest the involvement of a regulatory role of CASTOR1 in the AKT-mTORC1 loop.
[0196] AKT1 phosphorylation of CASTOR1 promotes RNF167-mediated ubiquitination and degradation of CASTOR1. Since these results suggested that the CASTOR1 protein level was strongly regulated by FBS, potentially through AKT activation, the mechanism mediating CASTOR1 degradation was examined. Consistent with the observed CASTOR1 protein level, FBS deprivation reduced CASTOR1 ubiquitination, while arginine deprivation had no noticeable effect (FIG. 1D). FBS re-stimulation after deprivation reversed the effect, restoring CASTOR1 ubiquitination, which was correlated with the reduced CASTOR1 ubiquitination and protein level (FIG. 7A). Together, these results confirmed that arginine did not significantly affect CASTOR1 protein level but FBS targeted CASTOR1 for ubiquitination and proteasome-dependent degradation.
[0198] To identify the E3 ubiquitin ligase(s) that might regulate CASTOR1 polyubiquitination and degradation, a panel of E3 ubiquitin ligases implicated in mTORC1 regulation was screened. Although ectopic expression of numerous E3 ubiquitin ligases decreased CASTOR1 protein level (FIG. 7D), only RNF167 increased CASTOR1 ubiquitination (FIG. 1F, and FIG. 7E). Consistently, knockdown of RNF167 decreased CASTOR1 ubiquitination and increased CASTOR1 protein level (FIGS. 1G and 1H) while overexpression of RNF167 decreased CASTOR1 protein level in a dose-dependent manner (FIG. 11 ). Neither overexpression nor knockdown of RNF167 had a notable effect on the CASTOR1 mRNA level (FIGS. 7F and 7G). Additionally, treatment with MG132 partially rescued RNF167-mediated downregulation of CASTOR1 protein (FIG. 7H). These results support a model that RNF167 targets CASTOR1 for ubiquitination and proteasome-dependent degradation (FIG. 1J).
[203] AKT1 phosphorylation of CASTOR1 at S14 enhances RNF167-targeting ubiquitination and degradation of CASTOR1 protein.
[0205] High CASTOR1 protein level overrides arginine activation of mTORC1:
Together, these results supported the notion that a high CASTOR1 protein level overrode arginine activation of mTORC1 at physiological conditions, and the mTORC1 activity was tightly regulated by CASTOR1 instead of the arginine status when CASTOR1 was expressed at a high level (FIG. 3H).
[0208] RNF167-mediated ubiquitination and AKT1-mediated phosphorylation of CASTOR1 promote breast cancer progression: the prognostic value of CASTOR1 mRNA expression in cancer was predicted using the TCGA database. Consistent with CASTOR1's inhibitory function on mTORC1 and tumor suppressive role, a lower CASTOR1 expression level was correlated with overall poor survival in pan-cancer analyses (FIG. 12A, 12B). At least 10 types of cancer showed a strong negative correlation including breast invasive carcinoma (BRCA), brain lower grade glioma (LGG), skin cutaneous melanoma (SKCM), head and neck squamous cell carcinoma (HNSC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), pancreatic adenocarcinoma (PAAD), glioblastoma multiforme (GBM) and acute myeloid leukemia (LAML) (FIG. 12C), of these, high RNF167 expression predicted a poor prognosis in GBM, LAML, SKCM, LGG and LIHC (FIG. 12D).
[0209] Breast cancer represents 12% of cancer diagnosed and is a major life threat for women in the United States. A high RNF167 expression level was found in breast tumors compared to the adjacent normal tissues (FIG. 12E). Furthermore, a lower CASTOR1 expression level (FIG. 12F, 12G) and a higher RNF167 expression level (FIG. 12H, 12I) were correlated with poor survival in ER+ and HER2+ breast cancer, respectively. In two pairs of ER+ and HER2+ breast cancer cell lines, an inverse correlation of activated AKT level with CASTOR1 protein level was identified (FIG. 13A). AKT interacted with CASTOR1 in MCF cells (FIG. 13B). Silencing of AKT1 and AKT inhibitor MK2206 enhanced exogenous and endogenous CASTOR1 protein levels in these cells, respectively (FIG. 13C, 13D). Consistently, overexpression of myr-HA-AKT1 but not the AKT kinase dead mutant K179M in MCF7 and T47D cells resulted in a dose-dependent reduction in CASTOR1 protein level (FIG. 13E, 13F).
[0210] Consistent with 293T cells, the affinity to exogenous and endogenous RNF167 was stronger for CASTOR1 S14D than WT and S14A in ER+ MCF7 and T47D cells, respectively (FIG. 14A-14C). Likewise, RNF167 overexpression decreased whereas RNF167 knockdown increased CASTOR1 protein level in MCF7 cells (FIG. 14D, 14E). Together these results indicated that similar to 293T cells, the CASTOR1 protein level was also regulated by AKT and RNF167 in breast cancer cells.
[0212] MCF7 cells that were transduced with a vector control, Flag-tagged CASTOR1 WT, S14A or S14D are subcutaneously engrafted into both flanks of nude mice. Ectopic expression of CASTOR1 WT and S14A significantly inhibited tumor growth in vivo, whereas S14D had a relatively less effect (FIGS. 5D-5F, and FIG. 16A). Additionally, mice injected with cells expressing CASTOR1 WT and S14A had higher survival rates than those of expressing vector control and S14D (FIG. 5G). Consistently, silencing of CASTOR1 in T47D cells promoted tumor growth in vivo and shortened the overall survival compared to a scrambled control group (FIGS. 5H-5J, and FIG. 16B). Taken together, these results revealed that AKT-mediated phosphorylation and RNF167-dependent ubiquitination led to a decreased CASTOR1 protein level in breast cancer cells, resulting in enhanced mTORC1 activation, cell proliferation, and tumorigenesis.
[0215] The mTORC1 pathway is often dysregulated in cancer, which is critical for the progression of cancer. While CASTOR1's mTORC1 inhibitory function is negated by arginine, a high level of CASTOR1 protein evades the effect of arginine and prevents arginine-mediated mTORC1 activation (FIG. 3). Furthermore, cancer cells often survive in an environment with low nutrients including a low level of arginine. Hence, it is expected that cancer cells would have evolved specific mechanisms to counter CASTOR1's inhibitory effect on mTORC1 in nutrients-deficient tumor microenvironment. In KSHV-transformed cells, KSHV-encoded miRNAs downregulate CASTOR1 to activate mTORC1. In other types of cancer, the AKT pathway is persistently activated as a result of mutation of AKT itself or its upstream pathways of growth factors3, which would phosphorylate CASTOR1 leading to its ubiquitination and degradation, and activation of mTORC1 regardless of the presence of high or low level of arginine (FIG. 17). Thus, cancer cells at least partially utilize constitutively active AKT to inhibit CASTOR1's function leading to constitutive mTORC1 activation.
[0216] While no consistent association of CASTOR1 mutation with any types of cancer has been identified so far, a lower mRNA expression level of CASTOR1 predicts a poor prognosis in 10 types of cancer (FIG. 12C). Importantly, a higher mRNA expression level of RNF167 predicts a poor prognosis in 6 of these 10 types of cancer (FIG. 12D). The fact that a low mRNA expression level of CASTOR1 and a high mRNA level of RNF167 predict a poor prognosis of these cancer types suggest the existence of an additional mechanism(s) regulating their mRNA expression. Pharmacological intervention of RNF167 leading to CASTOR1 activation could be considered as a potential therapeutic approach for these cancer types.
In summary, AKT1 phosphorylates CASTOR1 at S14 to enhance the already existing affinity of RNF167 to CASTOR1[Wingdings font/0xE0] RNF167 binding to CASTOR1 initiates RNF167 ubiquitination and degradation of CASTOR1 [Wingdings font/0xE0] CASTOR1 degradation initiates the tumorigenic mTORC1 cascade signaling. Increased CASTOR1 levels are associated with reduced mTORC1 signaling.
One cannot extrapolate the disclosure of the specification to the enablement of the claims because: (A) the specification does not provide a predictable correlation between the administration of RNF167 siRNA and treatment of cancer or breast cancer in a subject as broadly claimed, or for treatment of cancers expressing both RNF167 and CASTOR1 phosphorylated at Serine 14 (S14) (as recited in amended claim 3), and (B) the specification does not provide sufficient guidance for one of ordinary skill in the art to make and use any RNF167 siRNA homologous to any portion of any RNF167 nucleic acid sequence that predictably functions to treat cancer or breast cancer as claimed.
(A) The instant specification discloses that decreasing RNF167 levels by RNF167 knockdown increases CASTOR1 protein expression levels, and CASTOR1 is a tumor suppressor protein. However, the specification notes that some cancers, including breast cancers, can have low CASTOR1 expression levels with or without high RNF167 expression levels compared to normal cells, that are correlated to poor prognosis. Therefore, the RNF167 protein is not always overexpressed in cancers, its expression levels are not always associated with poor prognosis, and it appears poor prognosis is specifically dependent on CASTOR1 low expression levels. It is unclear what anti-cancer effects administering RNF167 siRNA has when administered to cancers already comprising low RNF167 expression levels (which is encompassed by the claimed cancers expressing RNF167), since it appears there are other cellular pathways contributing to tumorigenesis and poor prognosis when RNF167 expression is already low.
Further, the specification discloses that various conditions affect levels of CASTOR1 outside of RNF167, including presence and levels of FBS (mimicking nutrient-rich environment), AKT, arginine, and leucine. The presence of FBS (fetal bovine serum) in the cell culture allowed for ubiquitination and degradation of CASTOR1 protein (potentially through AKT activation), reducing CASTOR1 protein levels, while FBS deprivation resulted in higher CASTOR1 protein levels. Adding FBS to culture enhanced phosphorylation of CASTOR1 at S14 and CASTOR1 degradation, while FBS deprivation reduced phosphorylation of CASTOR1 resulting in relatively increased CASTOR1 protein levels. Chronic leucine or arginine deprivation activated AKT, reduced CASTOR1 mRNA and protein levels, and mTORC1 activity. Increasing levels of arginine activated mTORC1 tumorigenic signaling. Thus, numerous factors outside of RNF167 levels affect CASTOR1 protein levels, CASTOR1 phosphorylation at S14, and ubiquitination/degradation that enhance mTORC1 signaling and tumorigenesis, all adding to the unpredictability of simply administering RNF167 inhibitor or RNF167 siRNA to treat a disease or cancer.
The instant specification discloses: “These findings reveal a novel mechanism by which cancer cells overcome the suppressive effect of CASTOR1 in the nutrient-deficient tumor microenvironment, and hence identify a potential novel therapeutic target for treating mTORC1-associated diseases including cancer”; “these results revealed that AKT-mediated phosphorylation and RNF167-dependent ubiquitination led to a decreased CASTOR1 protein level in breast cancer cells, resulting in enhanced mTORC1 activation, cell proliferation, and tumorigenesis”; and “RNF167 overexpression decreased whereas RNF167 knockdown increased CASTOR1 protein level in MCF7 cells (FIG. 14D, 14E)” (see bold font sections cited above). Therefore, the instant specification indicates highly specific conditions must be present in the cancer for RNF167 inhibition to be successful in inhibiting cancer cells in vitro.
The specification does not provide any working examples of treating a disease or cancer in a subject, including cancers expressing CASTOR1 phosphorylated at S14 and expressing RNF167 at any levels, comprising administering a therapeutically effective amount of RNF167 inhibitor or siRNA to the subject.
In relevant art, CN111394461, Peng Xue teaches cervical cancer is characterized by decreased expression levels of RNF167 (English translation Example 1, p. 7-9; Figure 1 and 2; Table 1). Xue demonstrates that increasing the expression of RNF167 decreased cervical cancer cell proliferation (Example 2; section 8 Results; Figures 4 and 5). Peng teaches treating cervical cancer by administering treatment that increases the expression of RNF167 (claim 9; English translation p. 6). WO 2014/062720 also teaches administering an agent to treat cancer in a subject, wherein the agent induces or increases expression of cell cycle inhibition gene RNF167 ([51]; [54]; [125-127]) and wherein the cancer is breast cancer ([97]; [99]; [104]; claim 1). Wang et al (Molecular Cell, 2022, 82:770-784) teaches that RNF167 acts as a tumor suppressor. Wang demonstrates that increasing RNF167 expression in cancer cells results in suppressed anchorage-independent cell growth and proliferation (Figure S6E) while knockout of RNF167 expression in cells resulted in elevated mTORC1 activity and promoted anchorage-independent cell growth (Figures S6H and S6I; p. 776, col. 2). Van Dijk et al (Biochem. J. 2014, 459:27-36) teaches determining the function of mutant RNF167 proteins expressed in tumor cells in order to delineate the protein’s role in cancer, and concluded (p. 35, col. 2, last paragraph): “Our data suggest that a significant proportion of the RNF167/RNF13 mutations identified in human tumours cause a loss of function, leading to the notion that they may function as tumour suppressor genes.”
Therefore, the art teaches the opposite claimed. The art teaches increasing RNF167 expression in order to treat cancer, and that RNF167 acts as a tumor-suppressor. The art teaches that RNF167 knockout resulted in increased mTORC1 tumorigenic activity. Given the contradictory teaching of the art, and the various conditions of cell environment, AKT levels, arginine levels, and leucine levels demonstrated by the instant specification to affect CASTOR1 levels outside of RNF167 levels, a high quantity of experimentation would be required to determine what specific combination of conditions of a subject’s cancer must be present in order for an RNF167 inhibitor or siRNA to predictably treat cancer in a subject.
(B) The instant specification does not provide sufficient guidance on the manufacturing of RNF167 siRNA that predictably function to inhibit RNF167 and treat cancer, including what region of and percent homology with GenBank NM_015528.3 in order to be therapeutically effective in a subject. The instant specification discloses:
[0077] RNF167 is a RING-type E3 ligase involved in regulating protein trafficking, localization, and degradation by directly ubiquitinating targeted substrates. RNF167 is encoded by the RNF167 gene. In certain embodiments, RNF167 is a human RNF167 protein. In certain embodiments, RNF167 is a mouse RNF167 protein or a rat RNF167 protein.
[0078] In certain embodiments, RNF167 can be a human RNF167 protein having an amino acid sequence as set forth in GenBank Accession No. NP_056343 or an amino acid sequence at least about 95 percent or at least about 98 percent homologous thereto.
[0079] In certain embodiments, RNF167 can be an RNF167 protein that is post-translationally modified. For example, but not by way of limitation, the RNF167 protein can be glycosylated, e.g., at the N-terminus.
[0080] In certain embodiments, a nucleic acid encoding a human RNF167 protein of the present invention can comprise a nucleic acid sequence as set forth in GenBank Accession No. NM_015528.3 or a nucleic acid sequence at least about 95 percent or at least about 98 percent homologous thereto.
[0084] In certain embodiments, the RNF167 inhibitor can be an antisense nucleic acid, a shRNA, or a siRNA that is homologous to at least a portion of a RNF167 nucleic acid sequence. The homology of the portion relative to the RNF167 nucleic acid sequence can be at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 98 percent. The percent homology can be determined by, for example, BLAST or FASTA software. In certain embodiments, the homologous portion constitutes at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides. In certain embodiments, the antisense nucleic acid, shRNA, or siRNA molecules have up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 75, or up to 100 nucleotides in length. In certain embodiments, the antisense nucleic acid, shRNA, or siRNA molecules comprise DNA or atypical or non-naturally occurring residues, for example, but not limited to, phosphorothioate residues.
The specification discloses the RNF167 protein is not limiting, and can be derived from a mouse, rat, human, NP_056343, or an amino acid sequence at least 95% homologous thereto. It is noted that NP_056343 is “E3 ubiquitin-protein ligase RNF167 isoform a precursor [Homo sapiens],” a 350 amino acid sequence (see NCBI Reference Sequence: NP_056343.1, printed March 2026). Therefore, in most of the instant claims, the instantly claimed siRNA can be homologous to any RNF167 protein in the universe. With regards to amended claims 5 and 19, the specification does not provide guidance as to which portion of RNF167 GenBank Accession No. NM_015528.3 and what percent homology to which portion would predictably result in a therapeutically effective siRNA in a subject. The specification does not provide any working example of making an siRNA homologous to GenBank Accession No. NM_015528.3 that predictably therapeutically treats a subject having a disease or cancer when administered to the subject.
The state of the art indicates there are numerous isoforms and mutants of RNF167 that are still being discovered, as well as their unpredictable roles in cancer. Nair et al (bioRxiv772731, 2019) teaches they identified multiple isoforms of RNF167 of various lengths and sequences (Results, p. 11). Nair teaches RNF167 is normally a negative regulator of lysosomal exocytosis. Nair identified several tumor-associated RNF167 mutants, and determined after testing, that mutant K97N enhanced lysosomal exocytosis and plasma membrane resealing, a function that contributes to tumor progression (p. 5, 15, Figure 5 and 6; p. 17-19). Nair made RNF167 siRNA targeting two independent regions of 3’UTR region of RNF167 and transfected HeLa cells, where siRNA treatment resulted in a significant shift of lysosomes from the perinuclear region to the periphery of the cells, indicative of anterograde transport (Figure 3A; p. 13). RNF167 depletion resulted in an increased accumulation of lysosomes at the cell periphery (Figure 3C). Inhibition of RNF167 enhanced lysosomal exocytosis. Nair concludes that RNF167 functions normally to regulate the retrograde movement of lysosomes (p. 14-15) and some tumor-associated mutants of RNF167 result in enhanced lysosome accumulation at the cell periphery and exocytosis, a function that contributes to tumorigenesis (Discussion).
Van Dijk et al (Biochem. J. 2014, 459:27-36) also identified several tumor-associated mutant RNF167 sequences (Figure 1). Through experimentation, Van Dijk determined that some of the mutations result in ligase active proteins, but the proteins are mislocalized within the cells. Van Dijk determined that RNF167 mutants having loss-of-function PA of domains resulted in dominant-negative proteins, inhibiting the activity of wild-type RNF167 itself (abstract; p. 30, col. 2 to p. 33; Figure 2C and 5). Van Dijk teaches (p. 35, col. 2, last paragraph): “Our data suggest that a significant proportion of the RNF167/RNF13 mutations identified in human tumours cause a loss of function, leading to the notion that they may function as tumour suppressor genes.”
Zhang et al (International Journal of Nanomedicine, 2023, 18:7605-7635) reviews the unpredictable function of siRNA in the treatment of cancer. Zhang teaches (p. 7607):
Despite the potential of siRNAs in drug development, there are several extracellular and intracellular barriers that limit their application in vivo,49,50 especially in systemic delivery. The extracellular barriers include degradation of naked siRNAs by endogenous nucleases in serum,51 rapid renal clearance due to their small size and molecular weight,52 activation of the innate immune system triggered by siRNAs through Toll-like receptors (TLR)-dependent or TLR- independent pathways,53,54 plasma protein sequestration and entrapment by the reticuloendothelial system (RES),55–57 and membrane impermeability due to their negative charge and high hydrophilicity.58 The intracellular barriers mainly include endosomal entrapment59,60 and off-target effects.61
Zhang further teaches (p. 7613-7614):
Challenges in siRNA Drugs Development
Although siRNA offers significant advantages in cancer therapy, it is important to acknowledge the multitude of cancer- related genes that can be targeted using this approach. Nevertheless, the utilization of siRNA drugs in clinical cancer therapy encounters several obstacles that need to be overcome. The first challenge lies in the inherent instability of siRNAs under physiological conditions, making them susceptible to rapid clearance. As shown in Figure 3, the physiological milieu harbors a multitude of nucleases, leading to the degradation of siRNAs upon entry into the bloodstream.139 Furthermore, phagocytes are abundant in tissues and organs such as blood, liver, and spleen, particularly in the mononuclear phagocytic system (MPS)/reticuloendothelial system (RES), including mononuclear macrophages, phagocytic cells, and neutrophils, which engulf siRNAs entering the body.57 Moreover, significant amounts of siRNA are efficiently eliminated during their circulation to the kidneys by glomerular filtration.140 The unmodified siRNAs have a short half-life in blood, lasting only a few minutes to one hour.139 Even if siRNA successfully reaches the target cells, it initially encounters the endosomes and lysosomes, which have an acidic environment with a pH range of 5–6 and an even lower pH of 4.5 within the lysosomes. These compartments contain a number of nucleases capable of rapidly degrading siRNAs.141
The second challenge concerns the cellular uptake of free siRNAs. Due to its hydrophilic nature and negative charge, siRNA encounters obstacles when attempting to cross the negatively charged hydrophobic cell membranes and enter cancer cells, resulting in charge repulsion and impermeability.57
The third hurdle involves the escape of siRNAs from endosomes. The assembly of siRNA into the RISC complex for gene silencing can only occur in the cytoplasm. However, siRNA faces limitations in freely penetrating biological membranes, requiring strategies to achieve efficient endosomal escape and release into the cytoplasm, creating a barrier to siRNA delivery.57,141
The fourth obstacle involves the immunogenicity of siRNAs. Toll-like receptors (TLRs), which are pattern recognition receptors expressed on immune cells, can recognize pathogen-associated molecular patterns, including CpG DNA and viral dsRNA. TLR3, TLR7, and TLR8 specifically recognize nucleotide sequences in siRNAs, such as the UG dinucleotide and 5’-UGU-3’ motifs. Upon injection into the body, siRNAs activate the innate immune system, resulting in the production of substantial cytokines.54,142
The fifth challenge focuses on the off-target effects of siRNAs. Although siRNAs, with a length of 20 nucleotides, have a high specificity in terms of base-complementary pairing, cells contain numerous long-stranded mRNAs and miRNAs. Incomplete complementary pairing of siRNAs can lead to the degradation of unintended mRNAs and miRNAs, causing non-specific gene regulatory effects. Furthermore, cells express various miRNAs to regulate gene expression, and these miRNAs can have sequences identical to the target mRNAs. Consequently, siRNAs not only degrade target mRNAs but also miRNAs, leading to unpredictable changes in gene expression.143
Therefore, the art teaches the sequence of RNF167 proteins is highly variable, particularly in cancers, the different forms of RNF167 protein can result in different functions, rendering their role in cancers unpredictable, and the production of siRNA that successfully treat cancer is highly unpredictable and requires extensive experimentation to overcome the challenges outlined by Zhang. A high quantity of experimentation would be required to determine which RNF167 sequence to target with siRNA, and which siRNA sequence with any homology or at least 95% homology to any portion of GenBank Accession No. NM_015528.3 would predictably function to treat cancer as required by the claims.
Reasonable correlation must exist between the scope of the claims and scope of enablement set forth, and it cannot be reasonably predicted that any and all cancers or breast cancers will be treated by any and all RNF167 inhibitors or siRNA molecules as claimed.
Therefore, in view of the novel nature of the invention, the state of the art, the quantity of experimentation necessary, the breadth of the claims, lack of guidance in the specification, and the absence of working examples for treating diseases or cancers with the claimed RNF167 inhibitor or siRNA, it would require undue experimentation for one skilled in the art to practice the invention as claimed.
Response to Arguments
5. Applicants argue that claim 3 is amended and limited to treating cancers expressing CASTOR1 phosphorylated at S14 and RNF167. Applicants argue that the specification demonstrates treating cancer cells expressing CASTOR1 phosphorylated at S14 and RNF167 by knock-down of RNF167 that resulted in increased CASTOR1 levels. Applicants argue that the specification demonstrates that CASTOR1 expression inhibits tumor growth in vivo and enhances survival of mice in a xenograft model (Figures 5 and 16). Applicants argue this data enables the claimed invention.
6. The arguments have been considered but are not persuasive. Applicants are arguing limitations and data regarding limitations that are not recited in the claims. Claim 1 is not amended, therefore most of the claims still broadly encompass treating any disease or cancer in a subject with any RNF167 inhibitor. As stated in the rejection: “Given the contradictory teaching of the art, and the various conditions of cell environment, AKT levels, arginine levels, and leucine levels demonstrated by the instant specification to affect CASTOR1 levels outside of RNF167 levels, a high quantity of experimentation would be required to determine what specific combination of conditions of a subject’s cancer must be present in order for an RNF167 inhibitor or siRNA to predictably treat cancer in a subject.” Further, the instant specification does not provide a working example of administering an RNF167 inhibitor or siRNA to a subject that predictably results in treatment of a disease or cancer. In view of the unpredictable nature of the invention, the state of the art, the quantity of experimentation necessary, the breadth of the claims, lack of guidance in the specification, and the absence of working examples for treating diseases or cancers with the claimed RNF167 inhibitor or siRNA, it would require undue experimentation for one skilled in the art to practice the invention as claimed.
Regarding amended claim 3, claim 3 now recites that the disease treated is cancer that expresses CASTOR1 phosphorylated at serine 14 and RNF167. The claim encompasses cancers that express CASTOR1 phosphorylated at S14 at any level and express RNF167 at any level, including low expression and overexpression of either protein. As stated in the rejection, the state of the art contradicts the instant specification, teaching and demonstrating that increased RNF167 expression inhibits tumorigenesis, and RNF167 knockout resulted in increased mTORC1 tumorigenic signaling, concluding RNF167 is actually a tumor suppressor. The instant specification demonstrates that various conditions of cell environment, AKT levels, arginine levels, and leucine levels affect CASTOR1 levels outside of RNF167 levels. The instant specification fails to provide a working example of predictably treating cancer in a subject that expresses both CASTOR1 phosphorylated at S14 and RNF167 by administering an inhibitor of RNF167. Thus, even in cancers that express both CASTOR1 phosphorylated at S14 and RNF167, given the unpredictability of tumor environment and roles of RNF167 in tumor suppression and mTORC1 signaling, and given the lack of working examples of treatment, it cannot be predicted that such cancers in subjects will be treated by administration of an inhibitor of RNF167. Applicants have not argued against the tumor suppressor function of RNF167 demonstrated in the cited art.
7. Applicants argue that the specification provides enabling guidance for making and using an siRNA homologous to RNF167 to inhibit RNF167 and treat cancer. Applicants argue claims 5 and 19 are amended to recite the siRNA is homologous to a portion of RNF167 nucleic acid sequence GenBank Accession No. NM_015528.3. Applicants point to the decisions in Seagen Inc. v. Daiichi Sankyo Co. LTD and Amgen V. Sanofi and argue that some experimentation may be required to practice the claimed invention, and the enablement standard allows for "a reasonable amount of experimentation." Applicants argue that selection of siRNA in accordance with the amended claims would reflect reasonable experimentation as permitted under the enablement requirement. This is supported, for example, by detailed guidance and working examples provided in the specification. For example, a skilled person would be able to select siRNA as described in the specification at paragraphs [0083]-[0085], [0101]-[0102], and [0181- 0182] and Table 2 for use in the claimed method.
Applicants argue a skilled person would recognize that methods for designing siRNA are routine. Applicants argue Nair demonstrates validation of siRNA against RNF 167 (Nair at lines 222-231, 395-411, and FIG. S1D).
Applicants argue that the challenges discussed by Zhang for siRNA therapy do not relate to the design of siRNA sequences. In contrast, Zhang discusses unrelated challenges, for example: instability of siRNA under physiological conditions, making it susceptible to rapid clearance, obstacles in crossing cell membranes, release of siRNA from endosomes, immunogenicity, and off-target effects. Applicants argue that Examiner does not appear to argue that the claimed method lacks enablement in view of these challenges.
Applicants argue that while Zhang recognizes challenges in siRNA therapies, Zhang also identifies solutions that would be available to a skilled person. For example, siRNAs can be chemically modified to improve serum stability, extend half-life, enhance nuclease resistance, reduce off-target effects, and facilitate cellular uptake (Zhang at page 7615, fourth paragraph through page 7616, first paragraph). Various delivery systems (including viral systems, non-viral systems, lipid-based systems, polymer-based systems, siRNA-conjugate systems, and inorganic nanoparticle systems) are available to facilitate transport of siRNAs into target tissues and cells (Zhang at pages 7616- 7623). Bioresponsive materials (including pH-responsive biomaterials, redox-responsive biomaterials, enzyme-responsive biomaterials, hypoxia-responsive biomaterials, thermo-responsive biomaterials, and ATP-responsive biomaterials) can be used to control drug release (Zhang at pages 7623-7626).
8. The arguments have been considered but are not persuasive. Applicants are arguing limitations not recited in most of the claims. Claim 1 is not limited to siRNA as the RNF167 inhibitor, nor is it limited to siRNA that is homologous to a portion of RNF167 nucleic acid sequence GenBank Accession No. NM_015528.3. The claims broadly encompass administering any RNF167 inhibitor or RNF167 siRNA that is homologous to any RNF167 sequence derived from any species, mutant, or isotype of RNF167 and homologous to any portion thereof as small as two nucleotides. The cited art in the rejection demonstrates the genus of RNF167 sequences is vast and highly variable, and the specification fails to provide sufficient guidance that would direct one of skill in the art to the specific RNF167 inhibitor or siRNA sequence that would predictably treat a disease or cancer as required by the claims.
Regarding amended claims 5 and 19, the claimed siRNA is required to be homologous to any portion of GenBank Accession No. NM_015528.3, or have up to 5% sequence discrepancy in homology to a portion of GenBank Accession No. NM_015528.3. The instant specification discloses only two human RNF167 siRNA in Table 2 purchased from Sigma and identified by catalog number and not sequence. These siRNA were administered directly to in vitro cell culture to knock down RNF167 expression. No guidance is provided in the specification pointing to the critical sequence of GenBank Accession No. NM_015528.3 that the siRNA must be homologous to, and how many nucleotides it must be at least 95% homologous to, in order to predictably inhibit RNF167 and treat a disease or cancer in a subject.
Although Applicants argue that making and screening for inhibitory siRNA is routine, treating a disease or cancer in a subject in vivo with siRNA is not. The claims are not limited to the construction of an RNF167 siRNA, the claims require treating a disease or cancer comprising administering the siRNA to a subject. Examiner demonstrated the unpredictability in the art (citing Zhang) with regards to treating diseases or cancer in a subject by administering siRNA. Contrary to arguments, the challenges discussed by Zhang for siRNA therapy do relate to the design of siRNA sequences and therapeutics. Zhang teaches the design of therapeutically effective siRNA requires consideration of effective delivery and off-target effects (unintentional inhibition of other genes that hybridize to the siRNA), neither of which are addressed by the instant specification.
The specification lacks sufficient guidance for: (1) directing one to the critical sequence in GenBank Accession No. NM_015528.3 required for RNF167 siRNA homology to predictably inhibit RNF167 and therapeutically treat a disease or cancer; (2) directing one to the design of RNF167 siRNA that predictably allows for therapeutically effective delivery and avoids off-target effects in order to treat disease or cancer in a subject. There are no working examples demonstrating predictable treatment of subjects having a disease or cancer by administering an RNF167 siRNA. Even if an siRNA for RNF167 is screened for and identified, the cited art demonstrates there is significant unpredictability in the art for treating disease or cancer by administering siRNA because of the unpredictability in effective delivery and off-target effects. Applicants have not persuasively argued or demonstrated that the example of administering two commercial RNF167 siRNA directly to cells in culture reasonably and predictably extrapolates to the therapeutically effective treatment of disease or cancer in subjects in vivo, and to the therapeutic effects of any RNF167 siRNA as broadly claimed.
Although Applicants argue that Zhang offers some solutions to improve siRNA delivery and therapeutic effectiveness, these possible solutions suggested by Zhang reinforce the need for a high quantity of experimentation to find which solution would allow the RNF167 siRNA to predictably and therapeutically treat a disease or cancer as required by the claims. The instant claims and specification do not recite or disclose such solutions. Zhang establishes the state of the art is such that siRNA therapy is highly unpredictable and the instant specification does not provide a working example demonstrating that RNF167 siRNA predictably therapeutically treats a disease or cancer in a subject.
9. Conclusion: No claim is allowed.
Conclusion
10. THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
11. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642
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