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 .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 4/15/26 has been entered.
Applicant’s election without traverse of siRNA and R175 in the reply filed on 5/21/25 is acknowledged.
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.
Claims 1, 8, and 14 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.
The claims are directed to a method for inhibiting the growth of cancer, the method comprising administration to cancer cells that express one of the recited p53 mutants that promotes cancer growth of any siRNA that is targeted to any PEPD and disrupts association of the p53 mutant with the PEPD.
The specification does not adequately describe the structure required for the siRNA to be targeted to any location on any PEPD sequence and have the function of disrupting association of the p53 mutant with the PEPD.
The specification discloses that once incorporated into RISC, siRNA facilitate cleavage and degradation of targeted mRNA. Thus, for use in RNAi mediated silencing or downregulation of PEPD expression as described herein, siRNA, shRNA, or miRNA can be used (pages 10-11). This broad statement does not adequately describe the structure required for the siRNA to result in the recited function.
The specification discloses that PEPD (sequence undisclosed) binds to the recited p53 mutants in cells. The specification discloses that intratumoral injection of specific PEPD siRNA (sequences not disclosed in the example, so it is unclear what specific region of what specific PEPD target sequence is targeted by the siRNA) in specific breast cancer tumor types that comprise the instantly recited mutations strongly inhibited tumor growth (page 25). The examples do not specifically disclose which siRNAs were utilized. The specification discloses 4 siRNA single stranded sequences (SEQ ID NOs: 2, 3, 4, and 5) that have specific modification patterns in the sense strand that are identical to sequences in a specific location of a specific PEPD target sequence, which is not representative of the entire claimed genus of siRNAs.
The specification does not adequately describe the structure required between the siRNA and the target sequence in order to result in the recited function and the specification does not disclose the sequences of the siRNAs or the target.
The specification does not adequately describe the structure required for the function for any siRNA targeted to any PEPD to “disrupt an association of a p53 mutant with PEPD”. The minimal species of siRNAs targeting PEPD of the specification are not representative of the entire claimed genus and the specification does not even adequately describe the siRNAs or PEPD of the instant specification (the sequences are not disclosed). Without further description of the structure required for the function, one would not be able to readily recognize which siRNAs targeted to which PEPD sequences function by disrupting any type of association of a p53 mutant with PEPD. The specification does not adequately describe what structure or mechanism is required for the “targeting” of any PEPD.
Claim 1 has been amended to require for the disrupting the association results in the p53 mutant being freed from contact with the PEPD and the p53 mutant participates in killing of the cancer cells.
The specification does not adequately describe what structure is required for the siRNA to result in the disruption of the association of the p53 mutant and PEPD resulting in the p53 mutant being freed from contact with the PEPD and the p53 mutant “participates” in any manner in killing of the cancer cells.
The art does not define PEPD as being a single specific sequence and the claims are not even directed to any species (i.e. human) of PEPD. The claims encompass a method of introducing any siRNA “targeting” any PEPD, as well as encompass any PEPD homolog or allele from any species known or yet to be discovered of PEPD, as well as DNA genomic fragments, spliced variants or fragment that retains PEPD-like activity.
Furthermore, although the specification discloses siRNAs that are specific for a single PEPD sequence, the specification does not describe such agents directed to any other species of PEPD to describe the instantly claimed genus of any PEPD. One of ordinary skill in the art could not make such siRNAs targeted to any PEPD without knowledge of the target sequence and the structural relationship required between the siRNA and the sequence.
Additionally, the specification discloses 4 siRNA single stranded sequences (SEQ ID NOs: 2, 3, 4, and 5) that have specific modification patterns in the sense strand that are identical to sequences in a specific location of a specific PEPD target sequence, which is not representative of the entire claimed genus of siRNAs.
The MPEP states that for a generic claim, the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. See MPEP § 2163. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. See MPEP § 2163. Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad genus. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618. Additionally, in Carnegie Mellon University v. Hoffman-La Roche Inc., Nos. 07-1266, -1267 (Fed. Cir. Sept. 8, 2008), the Federal Circuit affirmed that a claim to a genus described in functional terms was not supported by the specification’s disclosure of species that were not representative of the entire genus. Furthermore, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
The claims are rejected under the written description requirement for failing to disclose adequate species to represent the claimed genus, the genus being which cancers express the instant mutations, which agents function as siRNAs targeting PEPD and having the function of disrupting any type of association of a p53 mutant with any PEPD.
The Guidelines for Examination of Patent Applications under the 35 USC § 112, first paragraph, “Written Description” Requirement”, published at Federal Register, Vol. 66, No. 4, pp. 1099-1111 outline the method of analysis of claims to determine whether adequate written description is present. The first step is to determine what the claim as a whole covers, i.e., discussion of the full scope of the claim. Second, the application should be fully reviewed to understand how applicant provides support for the claimed invention including each element and/or step, i.e., compare the scope of the claim with the scope of the description. Third, determine whether the applicant was in possession of the claimed invention as a whole at the time of filing.
With regards to siRNAs targeting PEPD, it appears that the structure is required to have an antisense strand that is fully complementary to 21-23 nucleotides of a specific target mRNA. For example, Elbashir et al. (The EMBO Journal, Vol. 20, No. 23, pages 6877-6888, 2001) teaches that duplexes of 21-23 nt RNAs are the sequence specific mediators of RNAi and that even single mismatches between the siRNA duplex and the target mRNA abolish interference (abstract and page 6888).
Additionally, Khvorova et al. (US 2008/0177051 A1) teaches that for siRNAs targeted to a specific target, some duplexes were active while others were not. FIG. 15 represents a typical screen of ninety siRNA duplexes (SEQ. ID NO. 0032-0120) positioned two base pairs apart. As the figure suggests, the functionality of the siRNA duplex is determined more by a particular sequence of the oligonucleotide than by the relative oligonucleotide position within a gene or excessively sensitive part of the mRNA, which is important for traditional anti-sense technology [0244].
Therefore, it was known in the art that not any siRNA that is targeted to a given mRNA will result in an active siRNA. The instant claims are directed to a method of delivering any siRNA targeted to any mRNA encoding prolidase that function by disrupting association of a p53 mutant with the PEPD, a genus of siRNAs that have not been adequately described in the specification.
The specification does not adequately describe the genus of siRNAs that would result in the recited functions.
Thus, having analyzed the claims with regard to the Written Description guidelines, it is clear that the specification does not disclose a representative number of species within the instant enormous genus that function as claimed. Thus, one skilled in the art would be led to conclude that Applicant was not in possession of the claimed invention at the time the application was filed.
Response to Arguments
Applicant argues: The specification provides adequate written description support for siRNAs targeting PEPD mRNA. The specification discloses that "siRNA are 21-23 nucleotide double-stranded RNA molecules that are recognized by the RNA-induced silencing complex (RISC). Once incorporated into RISC, siRNA facilitate cleavage and degradation of targeted mRNA." As-Filed Specification, page 10. The specification further discloses four specific PEPD siRNA sequences (SEQ ID NOs: 2, 3, 4, and 5) with specific modification patterns. See As-Filed Specification, page 11. These representative species demonstrate that Applicant was in possession of siRNAs targeting PEPD mRNA.
Disclosure in the specification of the general mechanism of siRNAs is not adequate to describe the instantly recited genus that are required to have a specific function. The four specific PEPD siRNA sequences of the specification are not representative of the entire claimed genus, which is directed to any species and encompasses a large genus of possible oligomers that would not have the structure to result in the recited function.
Applicant argues: Regarding the structure-function relationship, the specification clearly teaches that siRNAs targeting PEPD mRNA result in PEPD knockdown, which disrupts the association between PEPD and p53 mutants. The mechanism is described in detail: once PEPD is knocked down by siRNA, the p53 mutant is freed from contact with PEPD, undergoes K373 acetylation, refolds to wild-type conformation, and participates in killing cancer cells. See As-Filed Specification, Example 11. The specification demonstrates this mechanism with multiple p53 mutants, including RI 75H, R248Q, R273H, R280K, and E285A. See As-Filed Specification, Examples 1-12.
However, clearly not any siRNA targeting any PEPD mRNA would result in PEPD knockdown. Khvorova et al. (US 2008/0177051 A1) teaches that for siRNAs targeted to a specific target, some duplexes were active while others were not. FIG. 15 represents a typical screen of ninety siRNA duplexes (SEQ. ID NO. 0032-0120) positioned two base pairs apart. As the figure suggests, the functionality of the siRNA duplex is determined more by a particular sequence of the oligonucleotide than by the relative oligonucleotide position within a gene or excessively sensitive part of the mRNA, which is important for traditional anti-sense technology [0244].
Therefore, it was known in the art that not any siRNA that is targeted to a given mRNA will result in an active siRNA. The instant claims are directed to a method of delivering any siRNA targeted to any mRNA encoding prolidase that function by disrupting association of a p53 mutant with the PEPD, a genus of siRNAs that have not been adequately described in the specification.
Applicant argues: The specification adequately describes the claimed genus because the claims are limited to siRNAs targeted to PEPD mRNA, which is a known, defined target sequence. The four specific oligomers with specific modification patterns are not representative of the entire claimed genus.
Contrary to applicant’s argument, PEPD is not a single known, defined target sequence and the claims are not limited to any specific PEPD target sequence. The art does not define PEPD as being a single specific sequence and the claims are not even directed to any species (i.e. human) of PEPD. The claims encompass a method of introducing any siRNA “targeting” any PEPD, as well as encompass any PEPD homolog or allele from any species known or yet to be discovered of PEPD, as well as DNA genomic fragments, spliced variants or fragment that retains PEPD-like activity.
Furthermore, although the specification discloses siRNAs that are specific for a single PEPD sequence, the specification does not describe such agents directed to any other species of PEPD to describe the instantly claimed genus of any PEPD. One of ordinary skill in the art could not make such siRNAs targeted to any PEPD without knowledge of the target sequence and the structural relationship required between the siRNA and the sequence.
Additionally, the specification discloses 4 siRNA single stranded sequences (SEQ ID NOs: 2, 3, 4, and 5) that have specific modification patterns in the sense strand that are identical to sequences in a specific location of a specific PEPD target sequence, which is not representative of the entire claimed genus of siRNAs.
Claims 1, 8, and 14 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 a method of inhibiting PEPD via direct delivery of specific siRNA targeted to PEPD, does not reasonably provide enablement for a method of inhibiting the growth of any cancer that expresses any of the instantly recited p53 mutants that promotes cancer growth with any siRNA that is targeted to any region of any PEPD. 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 the invention commensurate in scope with these claims.
Factors to be considered in a determination of lack of enablement include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)
The claims are directed to a method for inhibiting the growth of cancer, the method comprising administration to cancer cells that express one of the recited p53 mutants that promotes cancer growth of any siRNA that is targeted to any PEPD and disrupts association of the p53 mutant with the PEPD.
The specification does not draw an adequate nexus between delivery via any means of any siRNA “targeted” to any location on any PEPD sequence and have the function of disrupting association of the p53 mutant with the PEPD.
The specification discloses that PEPD (sequence undisclosed) binds to the recited p53 mutants in cells. The specification discloses that intratumoral injection of specific PEPD siRNA (sequences not disclosed in the example, so it is unclear what specific region of what specific PEPD target sequence is targeted by the siRNA) in specific breast cancer tumor types that comprise the instantly recited mutations strongly inhibited tumor growth (page 25). The examples do not specifically disclose which siRNAs were utilized. The specification discloses 4 siRNA single stranded sequences (SEQ ID NOs: 2, 3, 4, and 5) that have specific modification patterns in the sense strand that are identical to sequences in a specific location of a specific PEPD target sequence, which is not commensurate in scope with the instantly recited genus of siRNAs.
The specification does not enable administration to cancer cells of any siRNA targeting any PEPD in any region with a predictable outcome of disruption of the association of the p53 mutant and PEPD resulting in the p53 mutant being freed from contact with the PEPD and the p53 mutant “participates” in any manner in killing of the cancer cells.
The specification discloses PEPD knockdown by siRNA may induce tumor suppressor activity of a specific p53 mutant, E285A, in BT-474 cells. PEPD knockdown in vitro resulted in marked decrease in nuclear p53 level, which is not commensurate in scope with the instant claims.
PEPD siRNA (undisclosed sequence in the example) strongly inhibited the growth of MDA-MB-231 (p53-R280K) breast cancer tumors (page 26).
Specification is not demonstrative of inhibiting the growth of any possible cancer with each of the recited mutations via delivery of any siRNA targeted to any PEPD sequence.
The specification discloses that PEPD binds to p53wT p53R1755H, p53R248Q, p53R273H, and p53R280K with almost identical affinity (see Fig. 6A).
The specification discloses screening of seven cell lines, including SKBR3 (homozygous p53R175H) HCC70 (homozygous p53R248Q),MDA-MB- 468 (homozygous p53R273H) MDA-MB-231 (homozygous p53R280K) MDA-MB-231 (p53KO),MCF-7 (p53WT), and CAL-51 (p53WT). They are human breast cancer cells, either HER2-positive (SKBR3), estrogen receptor-positive (MCF-7), or triple negative (all remaining cell lines). The specification discloses that all the p53 mutants are overexpressed, compared to p53WT, and p53 is absent in MDA-MB-231-p53KO, but interestingly, PEPD is also overexpressed in cells carrying a p53 mutant and even in MDA-MB-231-p53KO (Figure 6B).
The specification demonstrates intratumoral injection of PEPD siRNA strongly inhibit breast tumor growth with p53 R280K, p53 knockout, and p53 R175H. This is not commensurate in scope with a of inhibiting the growth of any cancer via delivery of any siRNA targeting any region of any PEPD.
The references cited herein illustrate the state of the art for therapeutic in vivo applications using dsRNA.
For example, Elbashir et al. (The EMBO Journal, Vol. 20, No. 23, pages 6877-6888, 2001) teaches that duplexes of 21-23 nt RNAs are the sequence specific mediators of RNAi and that even single mismatches between the siRNA duplex and the target mRNA abolish interference (abstract and page 6888).
Fujita et al. (Int. J. Mol. Sci. 2015, 16, 5254-5270) teach that two types of small RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), have a central function in RNAi technology. The success of RNAi-based therapeutic delivery may be dependent upon uncovering a delivery route, sophisticated delivery carriers, and nucleic acid modifications (page 5254). Fujita et al. teach that the success of an RNAi-based therapy in clinical trials rests on careful selection of target Int. J. Mol. Sci. 2015, 16 5263 genes and miRNAs. Moreover, we suggest that a delivery route, sophisticated delivery carriers, chemical modification, and modified RNAi platforms are needed to enhance RNAi effects in cancer cells (pages 5262-5263).
As outlined above, it is well known that there is a high level of unpredictability in the RNAi art for therapeutic in vivo applications regarding siRNAs. The scope of the claims in view of the specification as filed together do not reconcile the unpredictability in the art to enable one of skill in the art to make and/or use the claimed invention, namely a broad method of inhibiting cancer cell growth encompassing in vivo effects.
MPEP 2164.01
Any analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.
Also, MPEP 2164.01(a)
A conclusion of lack of enablement means that, based on the evidence regarding each of
the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed
invention without undue experimentation. In re Wright, 999 F.2d 1557,1562, 27
USPQ2d 1510, 1513 (Fed. Cir. 1993).
Given the teachings of the specification as discussed above, one skilled in the art could not predict a priori whether introduction of any siRNA targeting any region of any PEPD sequence in vivo by the broadly disclosed methodologies of the instantly claimed invention, would result in successful inhibition of cancer growth and disruption of the association of the p53 mutant with the PEPD sequence. To practice the claimed invention, one of skill in the art would have to de novo determine; the stability of the molecule in vivo, delivery of the molecule to the whole organism, specificity to the target tissue in vivo, dosage and toxicity in vivo, and entry of the molecule into the cell in vivo and the effective action therein. Without further guidance, one of skill in the art would have to practice a substantial amount of trial and error experimentation, an amount considered undue and not routine, to practice the instantly claimed invention.
A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention without undue experimentation (see MPEP 2164.01(a)).
Response to Arguments
Applicant argues: Notably, the Examiner acknowledges that the specification is enabling "for a method of inhibiting PEPD via delivery of siRNA targeted to PEPD." Office Action, page 8. The claims as previously amended are commensurate with this enabled scope-claim 1 recites administering "siRNA that is targeted to mRNA encoding prolidase (PEPD)." It is noted that the statement by examiner is specific to the siRNA that have been demonstrated to inhibit PEPD. The statement has been amended for clarity that the specification is enabling for a method of inhibiting PEPD via direct delivery of specific siRNA targeted to PEPD. However, the claims are not limited to such.
Applicant argues: The specification provides working examples demonstrating the claimed method works with multiple p53 mutants. The in vivo examples demonstrate that intratumoral injection of PEPD siRNA strongly inhibits tumor growth in breast cancer models with p53 R280K and R175H mutations. See As-Filed Specification, Example 12. Specifically, the specification teaches that "PEPD siRNA strongly inhibited the growth of MDA-MB-231 (p53R28K) tumors, and at the end of the experiment, average tumor size and tumor weight were only 10.6% and 8.6% of that in the control group respectively." As-Filed Specification, Example 12. Similarly, PEPD siRNA strongly inhibited growth of tumors expressing p53R175H Id
However, the claims are not limited to intratumoral injection of the PEPD siRNA of the specification to a subject with breast cancer with p53 R280K and R175H mutations.
The specification does not demonstrate inhibition of the growth of any possible cancer having one of the instantly recited mutations via direct delivery of any siRNA targeted to any region of any PEPD sequence.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
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.
Claim(s) 1, 8, and 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yang et al. (Nature Communications, 8, 2052, 2017, 1-15), in view of Suad et al. (Weizmann Institute of Science, Thesis, 2007, pages 1-17).
The references are considered as enabled as the instant specification.
Yang et al. teach: Eliminating PEPD causes cell death and tumor regression due to p53 activation. PEPD binds to the proline-rich domain in p53, which inhibits phosphorylation of nuclear p53 and MDM2-mediated mitochondrial translocation of nuclear and cytoplasmic p53. However, the PEPD-p53 complex is critical for p53 response to stress, as stress signals doxorubicin and H2O2 each must free p53 from PEPD in order to achieve robust p53 activation, which is mediated by reactive oxygen species. Thus, PEPD stores p53 for the stress response, but this also renders cells dependent on PEPD for survival, as it suppresses p53. This finding provides further understanding of p53 regulation and may have significant implications for the treatment of cancer and other diseases (abstract).
Yang et al. teach that PEPD suppression of p53 is essential for cell survival and tumor growth (page 2)
Yang et al. teach: PEPD knockdown markedly increases the transactivation activity of p53, which is consistent with modulation of various p53 target proteins by PEPD siRNA in a p53 dependent manner as described before. Moreover, in both cells lines, PEPD knockdown stimulated p53 phosphorylation (Fig. 4b) (page 5).
Yang et al. teach: Collectively, our results show that PEPD suppresses p53 transcriptional activity by inhibiting nuclear p53 phosphorylation in its transactivation domain (page 6).
Yang et al. teach: PEPD siRNA (Catalog number: SR303443; sequence: rCrGrA rArGrU rCrArA rCrArA rUrArC rCrArU rUrCrU rUrCA C), MDM2 siRNA #1(Catalog number: SR302849A; sequence: rCrCrC rUrArG rGrArA rUrUrU rArGrA rCrArA rCrCrU rGrAA A), MDM2 siRNA #2 (Catalog number: SR302849B; sequence: rGrUrA rCrUrA rGrArC rArArC rArUrG rUrArA rUrUrA rArUG A) (page 13).
Yang et al. teach: Disrupting PEPD suppression of p53 may be an important therapeutic strategy in cancer. We show that PEPD knockdown in tumors in mice causes p53 activation and tumor regression (page 11).
Yang et al. teach: In conclusion, PEPD sequester >50% cellular p53. Disrupting PEPD association with p53 frees p53 and unleashes its transcription-dependent and-independent functions, causing cell death and tumor regression. The PEPD-p53 complex is set up for robust p53 response to stress, and p53 separation from PEPD is a prerequisite for robust p53 activation by stress signals (page 12).
Yang et al. teach that to assess the role of PEPD in vivo, they generated subcutaneous tumors in nude mice using human bladder cancer cell line, UM-UC-3, and performed intratumoral injection of siRNA once every 3–4 days for four times. Tumors grew rapidly on control siRNA, but PEPD siRNA caused progressive tumor regression; at the end of the experiment, average tumor size and tumor weight in the PEPD siRNA group were only 17.3% and 17.1% of that in the control siRNA group (Fig. 1e, f). These results provide in vivo evidence that PEPD is essential for cell survival (page 4).
Yang et al. teach that p53 deletion of PRO was shown to have enhanced affinity for MDM2 and enhanced MDM2-mediated degradation.
Therefore, it would have been obvious to inhibit PEPD with a siRNA targeting PEPD to treat bladder cancer cells expressing p53.
Yang et al. do not teach that the p53 comprises a mutation of R175, R248, R273, R280, or E285.
However, Suad et al. teach that human p53 is a 393-residue protein that is mutated in more than 50% of invasive cancers. Suad et al. tech that the core domain is the target for nearly 96% of the known tumorigenic mutations in p53. Suad et al. teach that about 30% of all known mutations are found in six major “hotspots” residues (R175, G245, R248, R249, R273, and R282). Suad et al. teach that almost 3% of all known mutations are in codon 249 that is mutated in various cancer types in different organs mainly in the liver and lungs (page 8).
Given that the R175, G245, R248, R249, R273, and R282 mutations were known to be hotspots of tumorigenic p53 mutations, as taught by Saud et al., and it was known that siRNA targeted to PEPD results in disruption of PEPD association with p53 and freeing of p53 and unleashes its transcription-dependent and-independent functions, causing cell death and tumor regression, as taught by Yang et al., it would have been obvious to select an individual that has been diagnosed with a cancer that comprises cancer cells that express one of the known hotspot p53 mutations and deliver the siRNA of Yang et al. intratumorally with a reasonable expectation of the effects taught by Yang et al. (p53 activation and tumor regression, as well as disruption of PEPD association with p53 and freeing of p53 and unleashing its transcription-dependent and-independent functions) (claims 1, 8, and 14).
Conclusion
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/AMY ROSE HUDSON/Primary Examiner, Art Unit 1636
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