DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application Status
This action is written in response to applicant’s correspondence received 7/31/2023. Claims 1-22 are currently pending. Accordingly, claims 1-22 are examined herein.
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. 63/144,883, 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.
Claims 1 and 10 recite methods of reprogramming adult cochlea using an HDAC inhibitor and inhibitory nucleic acids targeting Fir, Mxi1, Fbxw7, and/or a combination thereof.
Claim Interpretation
Applicant defines an “inhibitory nucleic acid” as, “a nucleic acid, or a mimetic thereof, that when administered to a mammalian cell results in a decrease in the expression of a target gene.”(p. 13 ln 2-4). Per The Medical Biochemistry Page, there exist several mechanisms by which gene expression can be controlled in eukaryotes: alteration of chromatin structure, epigenetic control, transcriptional initiation, transcript processing, RNA transport, transcript stability (such as through degradation of target transcripts), translational intiation, post-translational modifications, protein transport, and control of protein stability (Regulation of gene expression. The Medical Biochemistry Page. Published 07/28/2020. Accessed via the Wayback Machine 01/28/2026. https://web.archive.org/web/20200728032222/https://themedicalbiochemistrypage.org/regulation-of-gene-expression/)
Based on the above information, the broadest reasonable interpretation of “inhibitory nucleic acids” encompasses the genus of nucleic acids which are capable of inhibiting the expression of a gene via any of the above mechanisms, e.g., through interfering with transcriptional initiation, transcript processing such as splicing, inducing mRNA degradation, inducing protein degradation, etc.. The broadest reasonable interpretation of “targeting” includes direct targeting as well as indirect, and therefore includes but is not limited to indirect mechanisms such as inhibition of transcription factors which regulate the expression of the recited genes. Because the claim does not specify whether it is the Mxi1/Fir/Fbxw7 DNA coding sequences, mRNAs, or proteins which are targeted, the claims also encompass inhibitory nucleic acids which can directly or indirectly inhibit any of those moieties.
According to Sridharan & Gogtay (Therapeutic nucleic acids: current clinical status. Br J Clin Pharmacol (2016) 82 659–672.), this genus encompasses antisense oligonucleotides (ASOs) that bind to and inhibit target mRNA; DNA aptamers that target coding nucleotides (p. 660 § Anti-sense oligonucleotides (ASOs) and DNA aptamers); siRNAs (p. 665 § RNA interference [RNAi] and short interfering RNAs [siRNAs]); microRNAs (p. 665 § MicroRNAs – their targeting and the use of antagonists and mimics of microRNA as therapeutic agents); RNA aptamers and RNA decoys that can bind and inhibit target nucleic acids or proteins (p. 667-668 §RNA aptamers and RNA decoys); ribozymes, which can act as enzymes to cleave mRNAs (p. 668 § Ribozymes); and circular RNAs, which can bind RNA-binding proteins to act as “sponges” (p. 668 § Circular RNAs). Collectively, these structures form a broad genus of nucleic acids with a variety of different structures and mechanisms of action.
In comparison to the breadth of the claims, the prior filed specification discloses methods of reprogramming adult cochlea using a combination of valproic acid, lithium chloride, forskolin, and siRNA against Mxi1 and Puf60 (Fir). However, the generic term “nucleic acid inhibitor” is absent from the priority documents, as is any mention of any of the other types of nucleic acid inhibitors known in the art and described above. Instead, they disclose only one specific type of inhibitory nucleic acid, which is not representative of the entire genus. Accordingly, the effective filing date of claims 1, 10 and their dependent claims is 02/02/2022.
Claim Rejections - 35 USC § 112(a)
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 1-3, 5-12, and 14-22 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.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
For the broadest reasonable interpretation of the claims, please refer to ‘Claim Interpretation’ above.
As discussed above and summarized here, claims 1 and 10 recite the term “inhibitory nucleic acids”, which broadly encompasses any type nucleic acid which has an inhibitory function against any form of Mxi1/Fir/Fbxw7, i.e., DNA, RNA or protein. These include siRNA, miRNA, antisense oligonucleotides, ribozymes, aptamers, RNA decoys, and circular RNAs.
In contrast to the breadth of the claimed inhibitory nucleic acids, the specification discloses working examples comprising siRNAs targeting Fir (siFIR, p. 28 ln 1), siRNA targeting Mxi1 (siMxi1,p. 28 ln 2), and/or siRNA targeting Fbxw7 (p. 28 ln 11). The specification does not disclose working examples involving any other type of structure of nucleic acid inhibitor. Nor does the specification disclose any embodiments in which other types of inhibitory nucleic acids are contemplated, or any functional characteristics alone or coupled with a known or disclosed correlation between structure and function.
The prior art shows that there is substantial structural and functional variety among inhibitory nucleic acids. For example, on page 447, in § Comparing ASOs and siRNAs, Crooke (Crooke et al. Antisense technology: an overview and prospectus. Nat Rev Drug Discov 20, 427–453 (2021).) discusses the two antisense technologies and their common requirement for the sequence of the target transcript, stating:
it is of interest to compare the two broadly established approaches that result in RNA target degradation AGO2-mediated and RNase-H1-mediated RNA degradation (Fig. 6). Each approach represents an antisense mechanism focusing on RNA targeting and employs chemically modified oligonucleotides, and so they share a number of attributes and concerns…They share a common theoretical objective: to alter disease phenotype by causing the selective degradation of target RNAs…Both approaches require the sequence of the target transcript to select appropriate sites — the receptor RNA sequences — to which the pharmacophore, the antisense oligonucleotide, binds.
Sridharan & Gogtay (cited above) further describe RNAi technology in general, stating:
RNAi is a process by which RNA molecules with sequences complementary to a gene’s coding sequence induce degradation of the corresponding messenger RNAs [mRNAs] thus blocking the translation of mRNA into protein….Therapy with siRNA thus has great potential application for diseases caused by abnormal expression or mutation such as cancers, viral infections and genetic disorders…as RNA interference can be experimentally triggered. (p. 665)
In summary, the prior art teaches that antisense oligonucleotides, siRNA, and miRNA are inhibitory nucleic acids which exert their inhibitory effect through complementary binding to a target transcript.
In contrast to antisense technologies for gene repression, other inhibitory nucleic acids have three-dimensional structures which correlate with a function of binding or degrading their targets. For example, ribozymes and DNAzymes are nucleic acids which form three-dimensional structures and are capable of catalytic activity, including RNA degradation. Zhang discloses an aptamer-DNAzyme conjugate for highly efficient in vivo gene silencing via RNA cleavage (Abstract)(Zhang et al. Y-Shaped CircularAptamer–DNAzyme Conjugates for Highly Efficient in Vivo Gene Silencing. CCS Chem. 2020, 2, 631–641.) Zhang notes that, “DNA-zymes hold strong enzymatic turnover properties and do not need to perform through the formation of the typical endogenic RNA-induced silencing complex (RISC).” (p. 632)”, and in Scheme 1 shows that the aptamer-DNAzyme conjugate folds into a complex three-dimensional structure. This shows that DNAzymes do not function through the same antisense base-pairing mechanisms as RNAi (siRNA, miRNA, ASOs), nor do they share a common structure.
Aptamers also have considerably variety in structure and function, and the correlation between their structure and function is not well-understood. Aptamers are known in the art which play a role in gene repression by binding transcription factors. For example, Xiang obtained a single stranded DNA aptamer that bound to the FOXM1 transcription factor and suppressed its transcriptional activities by binding FOXM1 on its consensus binding sites (Abstract, Xiang et al. Suppression of FOXM1 Transcriptional Activities via a Single-Stranded DNA Aptamer Generated by SELEX. Sci Rep 7, 45377 (2017).) Spöring describes aptamer-based gene switches for controlling gene expression, noting that, “During pre-mRNA processing, aptamers can regulate the splicing process, while at the stage of translation they may control translation initiation sequences or programmed ribosomal frame shifting. In addition, mRNA stability can be influenced through the accessibility of microRNA (miRNA) target sites or through the introduction of ligand-dependent ribozymes.” (Abstract, Introduction, Spöring et al. Aptamers in RNA-based switches of gene expression. Current Opinion in Biotechnology, Volume 63, 2020, Pages 34-40.). However, as Spöring also notes, “One major bottleneck in the development of artificial aptamer-based gene regulators is the low number of in vivo functional aptamer-ligand pairs…in vitro selection of aptamers poses the great challenge of identifying sequences that not only bind their designated ligand with high specificity and affinity but also enable riboswitch activity in vivo. In the past, many aptamer sequences have failed to function in vivo in RNA-based switch designs.” (p. 34 § Latest advances in aptamer selection via SELEX). What this indicates is that the relationship between aptamer structure and function was not well-understood in the art. This is supported by Ochoa & Milam (Direct Modeling of DNA and RNA Aptamers with AlphaFold 3: A Promising Tool for Predicting Aptamer Structures and Aptamer–Target Interactions. ACS Synthetic Biology 2025 14 (8), 3049-3064.), who state (p. 3050):
A significant barrier to aptamer optimization and rational design lies in the scarcity of structural data for functional nucleic acids, which limits understanding of nucleic acid folding…This imbalance of data makes it difficult to create generalizable models and fails to capture the full range of structural diversity possible in RNA and DNA molecules, particularly aptamers…Aptamers, despite their functional promise, remain largely underexplored, with just 117 DNA structures and 232 RNA structures reported in the PDB as of December 2024…Historically, computational tools for predicting aptamer structures have also faced limitations. Traditional secondary structure prediction algorithms… fail to consider noncanonical structures, such as G-quadruplexes, which arise from noncanonical nucleobase interactions (e.g., Hoogsteen base pairs). Furthermore, these algorithms can generate only 2D secondary structure
predictions and thus neglect 3D conformations entirely.
It is noted that Ochoa & Milam’s disclosures were published in 2025, which is after the effective filing date of the instant application. However, the disclosure is provided to evidence that the relationship between nucleic acid sequence and structure-binding function would not have been well-understood prior to the effective filing date, because it was still poorly understood several years later.
In summary, there is a high degree of structural and functional variation within the broad genus of inhibitory nucleic acids capable of directly or indirectly targeting expression of any given gene, including Mxi1, Fir and Fbxw7. The specification discloses only three publicly available siRNAs, but does not disclose the structures of any other inhibitory nucleic acids, such as ribozymes, aptamers, etc., nor does it disclose any correlation between structure and function for those types of inhibitory nucleic acids. While the specification does disclose the target gene sequences on pages 12-13, when taking into account the variability in structure of ribozymes and aptamers and the fact that the correlation between their structure and function is poorly understood, as well as the breadth of ways in which a target gene may be inhibited by an aptamer and/or ribozyme, as disclosed by Spöring, the disclosure of the target genes is not considered sufficient for the ordinary artisan to envision the genus of those inhibitory nucleic acids based solely on three species of siRNA. Therefore, one of ordinary skill in the art would conclude that based on the breadth of the claims, the limited amount of guidance provided by the specification and the art, the high degree of variation among members of the claimed genus, and further the unpredictability in the art, that one of ordinary skill in the art would conclude that Applicant was not in possession of the invention as broadly claimed.
Claims 4 and 13 are excluded from this rejection because they recite wherein the inhibitory nucleic acid is a siRNA, a shRNA, or antisense oligonucleotide. As discussed above, these types of inhibitory nucleic acids function via complementary base pairing to a target transcript, and the correlation between their structure and function is well-understood in the art. Therefore, given a target gene sequence, the ordinary artisan would be able to envision the genus of siRNAs, shRNAs or antisense oligonucleotides with sufficient complementarity to the target sequence that would render them capable of inhibiting expression of that gene.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
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.
Claims 1-4, 6, 8-13, 19 and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over WIPO Publication 2015/168149 A1 to Edge (of record, applicant’s submission, hereinafter ‘Edge’) in view of Samrajeewa (Samrajeewa et al. Therapeutic Potential of Wnt and Notch Signaling and Epigenetic Regulation in Mammalian Sensory Hair Cell Regeneration. Mol Ther. 2019 May 8;27(5):904-911.) and Yan (Yan et al. miR-155 contributes to the progression of glioma by enhancing Wnt/β-catenin pathway. Tumor Biol. (2015) 36:5323–5331.)
Regarding claim 1:
Edge teaches a method for reprogramming an adult mammalian inner ear for hair cell regeneration (i.e., modulating the epigenetic status and expression of certain genes to promote progenitor cell proliferation and hair cell differentiation) (see below; underlines added to emphasize the most relevant passages):
The present disclosure is based, at least in part, on the surprising discovery that epigenetic modulation results in supporting cell division and increases Atohl expression, which is expected to increase generation of hair cells and support cells. As shown herein, Sox2 and Pax2 interact with each other and the three prime (3 ') enhancer for Atohl, a transcription factor required for hair cell differentiation, at a compound consensus sequence, and these interactions lead to hair cell differentiation. Stimulation of the Wnt pathway also results in cell division and hair cell differentiation. In addition, modulating the epigenetic state of Atohl lead to an increase in Atohl expression. As increased Atohl leads to an increase in generation of hair cells, these epigenetic modifiers are expected to increase generation of hair cells from progenitors. (p. 6 ln 17-27)
The present disclosure provides that the epigenetic status of cochlear genes can
be modulated to promote sensory epithelial cell proliferation and hair cell differentiation. (p. 10 ln 8-10)
Edge teaches the method comprising: contacting an adult mammalian inner ear with an effective amount of a histone deacetylase (HDAC) inhibitor and one or more inhibitory nucleic acids targeting inhibitors of c-Myc and/or Wnt:
The compounds and methods described herein are appropriate for the treatment of mammalian (e.g., human) subjects who have or are at risk of developing hearing 25 disorders resulting from cochlear hair cell loss, preferably post-neonatal ( e.g., child, adolescent or adult, e.g., above the age of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 years) subjects. (p. 16 ln 23-7)
The methods include administering to the subject, e.g., to the inner ear of the subject, a
pharmaceutical composition comprising one or more of the following: a Histone Deacetylase (HDAC) inhibitor; a histone methyltransferase (HMT) inhibitor; a DNA
methyltransferase (DNMT) inhibitor; a Histone Lysine Demethylase (KDM) inhibitor;
an R-spondin; activators of c- and n-myc or Wnt agonists; and/or an inhibitory nucleic
acid that specifically reduces expression of Hic1. (p. 1 ln 27-29, p. 2 ln 1-4)
Hicl RNAi has been shown to increase the basal expression and Wnt-responsiveness of the Axin2 gene, which is another Wnt target. (p. 12 ln 12-14)
Downstream targets of the Wnt pathway, such as c-myc and n-myc, are mediators of many of the effects of Wnt signaling and are also useful targets for cochlear cell regeneration. Activators of c- and n-myc or Wnt agonists that increase the level of these proteins can therefore be used in the present methods as well, e.g., Wnt agonists…and inhibitors of Wnt inhibitors, e.g., interfering RNA (siRNA, shRNA) directed against DIckopf, WIF-1, shisa, kremen, SOST. sFRP, or axin. (p. 12 ln 25-31)
Edge claims the method comprising administration of HDAC inhibitors (claims 1-3) and provides working examples showing that HDAC inhibitors increase expression of Atoh1 and would be expected to lead to generation of new hair cells (Fig. 15, Example 10). Edge also claims the method comprising administration of anti-Hic1 siRNA and shows that reducing Hic1 activity results in a dramatic increase in Atoh1 expression (claims 13-14, 16, Fig. 17A-B, Example 11). While Edge does not provide a working example of administering both of these elements together, Edge’s teachings that the combination of HDAC inhibitors and Wnt activators are effective at reprogramming hair cells in the adult mammalian inner ear are supported by Samrajeewa, which describes the successful application of the approach in adult mice and human inner ear tissue:
There is a growing consensus that combinational strategies may be able to synergistically enhance the regenerative capacity in neonatal and adult cochleae. For example, the in vitro hair cell yield from Lgr5-positive cells isolated from neonatal mice and grown as organoids can be further improved by treatment with the Wnt activator CHIR99021 (CHIR) and the histone deacetylase (HDAC) inhibitor valproic acid (VPA; which is thought to enhance histone H3K9 acetylation)…These findings were successfully reproduced in Lgr5-positive cells isolated from adult mice, as well as non-human primates and healthy human inner ear tissue. (p. 907 §Combinational Strategies for Hair Cell Regeneration)
Samrajeewa also notes that “there are no biological therapies for hearing loss” (Abstract), and that, “Although modulation of signaling pathways can promote hair cell regeneration in the neonatal cochlea, this regenerative response is lost or limited in adult stages…Use of Wnt and Notch modulators in combination with drugs targeted against chromatin-remodeling enzymes, such as HDAC inhibitors…highlights the positive effects that altering access to target genes can have on the regenerative response of mature inner ear tissue.” (p.907). Therefore, Samrajeewa establishes that there was a known and largely unmet need in the art for therapies for hearing loss in adult subjects, and further suggests combined therapies using Wnt modulators and HDAC inhibitors as a solution.
In summary, Edge teaches a method of reprogramming an adult mammalian inner ear for hair cell regeneration, the method comprising administration of a HDAC inhibitor in combination with a Wnt activator (i.e., RNAi agents against c-myc, n-myc and/or Wnt inhibitors, such as Hic-1). Edge provides a teaching, suggestion or motivation to use RNAi agents against c-myc and/or Wnt inhibitors with a HDAC inhibitor. Samrajeewa supports Edge’s teachings, providing the ordinary artisan with additional reasonable expectations of success by further showing that the combination of HDAC inhibitors and Wnt activators was known to be effective at reprogramming hair cells in the adult mammalian inner ear, and provides a motivation to pursue these types of combinational strategies by stating that they may synergistically enhance the regeneration of adult inner ear hair cells.
While Edge teaches the method comprising administration of nucleic acid inhibitors of Hic-1, itself a known inhibitor of the Wnt pathway, Edge does not teach the method comprising administration of nucleic acid inhibitors of Mxi1.
Yan teaches a nucleic acid inhibitor of Mxi1, miR-155, and that miR-155 activates the Wnt pathway:
miR-155 contributes to the progression of glioma by enhancing Wnt/β-catenin pathway (Title)
The suppression of miR-155 attenuated the proliferation of glioma cells and the activation of Wnt pathway…miR-155 level was inversely correlated with the abundance of HMG-box transcription factor 1 (HBP1), a strong Wnt pathway inhibitor…miR-155 promoted the progression of glioma by enhancing the activation of Wnt pathway. (Abstract)
miR-155 promoted the proliferation and invasion of glioblastoma cells through suppressing…MXI1 (p. 5330 §Discussion)
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention, absent evidence of secondary considerations of non-obviousness, to have combined the method of reprogramming adult inner ear hair cells via administration of an HDAC inhibitor and c-myc and/or Wnt-pathway-activating RNAi, as taught by Edge and supported by Samrajeewa, with Yan’s teachings that administration of a nucleic acid inhibitor of Mxi1, miR-155, enhanced activation of the Wnt pathway. The ordinary artisan would have been motivated by Edge’s teachings regarding combined HDAC and RNAi therapies/Wnt activators to seek out RNAi agents for activating Wnt (by targeting Wnt inhibitors for silencing). This motivation would have been encouraged by Samrajeewa’s teachings that such combinational therapies using Wnt activators and HDAC inhibitors together are more effective for inducing regeneration of hair cells in the adult inner ear than either one alone, and further that there was a recognized need for more such therapies. The search for additional RNAi agents for activating Wnt would have led them to Yan, which teaches that miR-155 (a nucleic acid inhibitor of Mxi1) is a Wnt activator, and would thus have been suitable for use in the therapies suggested by Edge and Samrajeewa. Based on the combination of those teachings, the ordinary artisan would have had a reasonable expectation that a combinational strategy combining a known HDAC inhibitor with a known Wnt-activating nucleic acid inhibitor, miR-155, would have successfully induced hair cell proliferation and regeneration (i.e., reprogramming) in the adult inner ear.
Regarding claims 10 and 21-22, Edge teaches the same methods for treating vestibular dysfunction in a human subject having hearing loss (hearing problems) from vestibular dysfunction (see p. 18 ln 1-8), the method also comprising administration of an Atoh1 activator (Sox2 and Pax2 cDNA; see FIG. 5 and p. 29 ln 27-30, which describe increased activation of Atoh1 by administration of “equal amounts of Pax2 and Sox2 cDNA.)
Regarding claims 2-3 and 11-12, Edge teaches wherein the HDAC inhibitor is valproic acid (see Edge claim 3).
Regarding claims 4 and 13, Edge teaches wherein the nucleic acid inhibitor is a siRNA (see above).
Regarding claims 6 and 19, Edge teaches administration of Wnt agonists (see above).
Regarding claim 8, Edge teaches that expression of Sox2 is an inherent characteristic of undifferentiated progenitor inner ear stem cells:
Both Pax2 and Sox2 are expressed during development of the otocyst and are important for the development of the cochlea during tissue morphogenesis. Pax2 and Sox2 are both expressed in inner ear stem cells…Their level of expression decreases when inner ear stem cells are transferred from a proliferative self-renewing culture (as floating neurospheres in growth factors) to a differentiating culture (p. 23 ln 31-33, p 24. ln 1-4)
Regarding claim 9, Edge teaches wherein the contact occurs in the inner ear of the subject (e.g., claims 1, 4).
Claims 1, 5, 10 and 14-18 are rejected under 35 U.S.C. 103 as being unpatentable over Edge, Samrajeewa and Yan, as applied to claims 1-4, 6, 8-13, 19 and 21-22 above, further in view of Cukier (Cukier eta l. Molecular basis of FIR-mediated c-myc transcriptional control. Nat Struct Mol Biol. 2010 Sep;17(9):1058-64.)
Edge, Samrajeewa and Yan render obvious the methods of reprogramming inner ear hair cells/treating a human subject having vestibular dysfunction, comprising administering a composition comprising an HDAC inhibitor, Mxi1 nucleic acid inhibitor, and an Atoh1 activator to upregulate Atoh1 expression, as recited in claims 1 and/or 10, from which claims 5 and 14 depend, as discussed above.
Edge, Samrajeewa and Yan do not teach administration of inhibitory acids targeting Fir, as recited in the alternative in claims 1 and 10 and required by claims 5 and 14-15.
However, Edge does teach compositions comprising nucleic acid inhibitors for downregulation of c-myc inhibitors (i.e., activation of c-myc). Additionally, Samrajeewa teaches therapies for hearing loss which comprise administration of an Atoh1 adenoviral expression vector to adult human subjects, and that the combination of this approach with HDAC inhibition and Wnt activation can produce a more robust regenerative response:
Novartis Pharmaceuticals is currently conducting a clinical trial on the use of CGF166, a recombinant adenovirus 5 (Ad5) vector encoding the human Atonal transcription factor, in patients with severe-to-profound bilateral hearing loss for safety, tolerability, and changes in vestibular and auditory functions. (p. 906)
In addition to VPA, the combination of Wnt activation with other forms of pharmacological and genetic manipulation can also produce a more robust regenerative response in the neonatal cochlea. For instance, the combination of b-catenin stabilization with ectopic Atoh1 expression in vivo in Lgr5-positive cells of the neonatal cochlea promotes a synergistic increase in supporting cell proliferation and hair cell induction (p. 907)
Cukier teaches that FIR shuts off transcription of c-myc via its interactions with FUSE and the FUSE-binding protein (FBP):
The far upstream element (FUSE) upstream of the c-myc promoter mediates a fast transcription-responsive mechanism responsible for an upsurge of c-Myc levels during the cell cycle…FUSE-based c-Myc upregulation is mediated by the unwinding and opening of an AT-rich stretch (the FUSE sequence) located about 1.7 kb upstream of the c-myc promoter…The FUSE DNA noncoding strand (henceforth referred to as ssFUSE or ssFUSE DNA) recruits the FUSE-binding protein (FBP), which in turn interacts with transcription factor IIH (TFIIH)…and increases the rate of productive c-myc transcription…The FBP–FUSE complex recruits the FBP-interacting repressor (FIR) protein that interacts with both FBP and ssFUSE DNA. Upon binding FBP and ssFUSE, FIR interacts with TFIIH7, reducing FBP-mediated transcription (p. 1058)
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have combined the method of reprogramming inner ear hair cells/treating vestibular dysfunction via a combinational therapy comprising a combination of HDAC inhibitors, c-myc and/or Wnt pathway activators such as a nucleic acid inhibitor of Mxi1, and Atoh1 expression vectors, as taught by Edge, Samrajeewa and Yan, with Cukier’s teachings that FIR is a transcriptional repressor of c-myc. The ordinary artisan would have recognized that FIR offered a target for RNAi to activate c-myc, as suggested by Edge, and would have been motivated to add it to the pre-existing combinational therapy to produce a more robust regenerative response.
Claims 7 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Edge, Samrajeewa and Yan, as applied to claims 1-4, 6, 8-13, 19 and 21-22 above, further in view of Xia (Activation of Wnt/b-catenin signaling by lithium chloride attenuates D-galactose-induced neurodegeneration in the auditory cortex of a rat model of aging. FEBS Open Bio 7 (2017) 759–776.)
Edge, Samrajeewa and Yan render obvious the methods of reprogramming inner ear hair cells/treating a human subject having vestibular dysfunction, comprising administering a composition comprising an HDAC inhibitor, Mxi1 nucleic acid inhibitor, and an Atoh1 activator, as recited in claims 1 and/or 10, from which claims 5 and 14 depend, as discussed above.
Edge further teaches the method comprising administration of Wnt agonists (see above).
Edge does not teach that the Wnt agonist is lithium chloride.
Xia teaches that lithium chloride activates Wnt and attenuates age-related hearing loss (Title, Abstract).
It would have been obvious to combine the combinational method of reprogramming inner ear hair cells/treating a human subject having vestibular dysfunction, as taught by Edge, Samrajeewa and Yan, by adding a further small molecule Wnt activator to achieve more robust Wnt activation for improved regeneration in adult hair cells. The ordinary artisan would have a reasonable expectation of success based on Xia’s teachings that lithium chloride was a known effective Wnt agonist, combined with Edge’s teachings that Wnt agonists are effective in such therapies when combined with HDAC inhibitors, and Samrajeewa’s suggestion to pursue combinational therapies.
Claims 1-4, 6, 8-13, 19 and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over WIPO Publication 2015/168149 A1 to Edge (of record, applicant’s submission, hereinafter ‘Edge’) in view of Samrajeewa (Samrajeewa et al. Therapeutic Potential of Wnt and Notch Signaling and Epigenetic Regulation in Mammalian Sensory Hair Cell Regeneration. Mol Ther. 2019 May 8;27(5):904-911.) and Jiang (Jiang et al. Tumor suppressor Fbxw7 antagonizes WNT signaling by targeting β-catenin for degradation in pancreatic cancer. Tumor Biol. (2016) 37:13893–13902.)
Regarding claim 1:
Edge and Samrajeewa teach the limitations of claims 1-4, 6, 8-13, 19 and 21-22 as far as they pertain to the method comprising an inhibitory nucleic acid targeting Mxi1, a known inhibitor of the Wnt pathway. Edge and Samrajeewa also suggest combining HDAC inhibitors with agents to activate the c-myc and/or Wnt pathways, including agonists and inhibitory nucleic acid molecules targeting inhibitors of those pathways.
Edge does not teach the method comprising administration of nucleic acid inhibitors of Fbxw7.
Jiang teaches that Fbxw7 is a Wnt antagonist, and that RNAi knockdown of Fbxw7 activates both c-myc and Wnt (see Abstract). Jiang also teaches a specific short hairpin RNA inhibitor of Fbxw7 (p. 13894 § Fbxw7 and β-catenin knockdown).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention, absent evidence of secondary considerations of non-obviousness, to have combined the method of reprogramming adult inner ear hair cells via administration of an HDAC inhibitor and c-myc and/or Wnt-pathway-activating RNAi, as taught by Edge and supported by Samrajeewa, with Yan’s teachings that administration of a nucleic acid inhibitor of Fbxw7 enhanced activation of both the c-myc and Wnt pathways. The ordinary artisan would have been motivated by Edge’s teachings regarding combined HDAC and RNAi therapies/c-myc and/or Wnt activators to seek out RNAi agents for activating c-myc and/or Wnt (by targeting c-myc and/or Wnt inhibitors for silencing). This motivation would have been encouraged by Samrajeewa’s teachings that such combinational therapies using Wnt activators and HDAC inhibitors together are more effective for inducing regeneration of hair cells in the adult inner ear than either one alone, and further that there was a recognized need for more such therapies. The search for additional RNAi agents for activating c-myc and/or Wnt would have led them to Jiang, which teaches a shRNA inhibitor of Fbxw7, and further teaches that silencing of Fbxw7 activates those pathways, and would thus have been suitable for use in the therapies suggested by Edge and Samrajeewa. Based on the combination of those teachings, the ordinary artisan would have had a reasonable expectation that a combinational strategy combining a known HDAC inhibitor with a known shRNA inhibitor of Fbxw7, with a known ability to activate Wnt and c-myc via Fbxw7 knockdown, would have successfully induced hair cell proliferation and regeneration (i.e., reprogramming) in the adult inner ear.
Regarding the other claims named in the rejection, Edge teaches those limitations as already discussed above.
Claims 7 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Edge, Samrajeewa and Jiang, as applied to claims 1-4, 6, 8-13, 19 and 21-22 above, further in view of Xia (Activation of Wnt/b-catenin signaling by lithium chloride attenuates D-galactose-induced neurodegeneration in the auditory cortex of a rat model of aging. FEBS Open Bio 7 (2017) 759–776.)
Edge, Samrajeewa and Jiang render obvious the methods of reprogramming inner ear hair cells/treating a human subject having vestibular dysfunction, comprising administering a composition comprising an HDAC inhibitor, Fbxw7 nucleic acid inhibitor, and an Atoh1 activator, as recited in claims 1 and/or 10, from which claims 5 and 14 depend, as discussed above.
Edge further teaches the method comprising administration of Wnt agonists (see above).
Edge does not teach that the Wnt agonist is lithium chloride.
Xia teaches that lithium chloride activates Wnt and attenuates age-related hearing loss (Title, Abstract).
It would have been obvious to combine the combinational method of reprogramming inner ear hair cells/treating a human subject having vestibular dysfunction, as taught by Edge, Samrajeewa and Yan, by adding a further small molecule Wnt activator to achieve more robust Wnt activation for improved regeneration in adult hair cells. The ordinary artisan would have a reasonable expectation of success based on Xia’s teachings that lithium chloride was a known effective Wnt agonist, combined with Edge’s teachings that Wnt agonists are effective in such therapies when combined with HDAC inhibitors, and Samrajeewa’s suggestion to pursue combinational therapies.
Relevant Art Cited
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure for the reasons that follow.
Zhou (Zhou et al. MicroRNA-155 Promotes Glioma Cell Proliferation via the Regulation of MXI1. (2013) PLoS ONE 8(12):e83055.)
Zhou teaches that miR-155 downregulates Mxi1, confirms that Mxi1 is a c-myc inhibitor, and that miR-155 activates c-myc in addition to Wnt (see Abstract). This reference is relevant because Edge suggests the combination of HDAC inhibitors with both c-myc and Wnt activators to reprogram inner ear hair stem cells, and Zhou teaches that miR-155 would have been able to activate both targets.
Conclusion
No claim is allowed at this time.
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/A.M.Z./Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636