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 .
Office Action: Notice
Any objection or rejection of record in the previous Office Action, mailed 8/11/2025,
which is not addressed in this action has been withdrawn in light of Applicants' amendments
and/or arguments. This action is FINAL.
Claim Status
Claims 19, 21, 27, 28, and 30-50 were cancelled (11/15/2022). Claims 2, 8, 11, 13, 51-52, 55-56 and 58 have been amended (12/11/2025). Claims 1-18, 20, 22-26, 29, and 51-60 are under examination (12/11/2025).
Priority
Claims 1-18, 20, 22-26, 29, and 51-60 receive a priority date of 11/23/2019, the effective filing date of US Provisional Patent 62/939,599.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure
statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information
submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be
incorporated into the specification but must be submitted in a separate paper." Therefore, unless
the references have been cited by the examiner on form PTO-892, they have not been
considered.
The information disclosure statement (IDS) submitted on 12/11/2025 is being considered by the examiner.
Objections Withdrawn
Specification:
The objections to the specification due to the use of a trademark or tradenames are withdrawn in view of Applicant’s amendments.
Claims:
The objections to claim 8 for minor clerical issues are withdrawn in view of Applicant’s amendments of claim 8.
Rejections Withdrawn
Claim Rejections - 35 USC § 101
The rejection of claim 51 under 35 U.S.C. 101 is withdrawn in view of Applicant’s amendments of claim 51, as well as Applicant’s clarification of the intended invention. Specifically, the Applicant amended claim 51 to recite an isolated cell, which removes the claim from encompassing a human organism under AIA 33(a). As acknowledged in the Office Action (8/11/2025), reciting an “isolated cell” is remedial and places the claim within statutory subject matter. An isolated cell, even if of human origin, does not constitute a human organism or an integrated component of the human body for purposes of § 101. Accordingly, the amended claim is directed to a statutory composition of matter.
Claim Rejections - 35 USC § 112(b)
The rejections of claims 52-60 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, are withdrawn in view of Applicant’s amendments of claims 52, 55-56, and 58 to address and further clarify indefiniteness.
Claim Rejections - 35 USC § 102
The rejection of claims 55-60 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Hovnanian et al., (WO 0164747 A1, published 9/7/2001) is withdrawn in view of Applicant’s amendments of claims 55-56.
Rejections Maintained
Claim Rejections - 35 USC § 102
Claims 1-11, 14, 17-18, 20, 22-23, 29, and 51-54 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Hovnanian et al., (WO 0164747 A1, published 9/7/2001). Rejection has been modified to reflect Applicant’s amendments (12/11/2025).
Regarding claim 1, Hovnanian teaches an invention providing a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINK5 gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and
screening for expression of the reporter gene product (p. 27, lines 1-10). Specifically, Hovnanian teaches that the above method of the invention can be used to identify compounds which up-regulate the expression of SPINK5 and hence have potential pharmacological activity in the treatment of Netherton's disease or atopic disease, including a promoter region or spacer of the human SPINK5 gene (i.e., sequences immediately upstream of the LEKTI transcription start site which are necessary for correctly positioning RNA polymerase and also to the proximal promoter region plus any additional sequence elements which may be involved in regulating LEKTI gene expression (p. 27, lines 20-30). Specifically, Hovnanian teaches that the LEKTI fusion proteins will typically be made by recombinant nucleic acid techniques in which two or
more open reading frames that are translationally fused or may be chemically synthesized
for purification and thus creating a shift in the reading frame (p. 21, lines 30-40).
Hovnanian also teaches that the previously described method can be used to target specific regions for mutational analyses, including sites of mononucleotide repeats (Example 2; p. 33, lines 25-30). Specifically, Hovnanian teaches that sites of mutational analyses are typically chosen based on physical proximity to a marker shown to be associated with atopic disease
but which have not formally been shown to be associated with atopic disease; including "linkage disequilibrium" which occurs between a marker polymorphism (i.e., a DNA
polymorphism which is 'silent') and a functional polymorphism (i.e., genetic variation which affects phenotype or which contributes to a genetically determined trait) if the marker is situated in close proximity to the functional polymorphism (i.e., polyguanine, polycytosine) (p. 4, lines 1-10).
Regarding claims 2-3, Hovnanian teaches that the previously described method of analysis via a nucleotide construct includes mutational analyses targeted based on mutations/variants occurring in an intron as a certain number of nucleotides + or – the nucleotide at the nearest exon/intron boundary (p. 18, lines 1-10). Further, Hovnanian teaches that these specified introns can range between 5-50 bp or contain individualized guanines or cytosines (Table 6), as well as specified mutational patterns or transitions within SPINK5 (i.e., C22) (Table 1a; p. 17, lines 1-10).
Regarding claims 4-11, Hovnanian teaches that the previously described method of analysis via nucleotide construct incorporates oligonucleotide molecules or spacers that typically range from 15 to 50 nucleotides in length and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art (p. 14, lines 25-35). Further, Hovnanian teaches that these markers can include
a gene encoding a dominant selectable marker, such as neomycin phosphotransferase for versions A and B (p. 29, lines 10-15; p. 43, lines 35-40). Further, these Myc-tags (i.e., Myc-epitope) can incorporate fusion with the carboxy-terminal end for immunopurification or immunodetection of the expressed polypeptide to act as an immunoreporter (p. 44, lines 25-30) or alternatively for purification, where the heterologous protein or peptide tag commonly being removed after purification by chemical or enzymatic cleavage (p. 21, lines 30-40).
Specifically, Hovnanian teaches that the ORFs, included in this previously described method, can also be specifically labeled via reporter genes which may be used in accordance
with the invention include those which encode a fluorescent product, such as green fluorescent protein (GFP) or other autonomous fluorescent proteins of this type (p. 28, lines 5-10).
Regarding claim 14, Hovnanian teaches that the previously described method of analysis via a nucleotide construct includes mutational analyses includes the loss-of-function mutation which may be a nonsense mutation (i.e., a single nucleotide changes leading to the incorporation of a premature translation termination codon), a frameshift mutation (i.e., an insertion or deletion) (p. 8, lines 10-15).
Regarding claim 17, Hovnanian teaches that sites of mutational analyses are typically chosen based on physical proximity to a marker shown to be associated with atopic disease
but which have not formally been shown to be associated with atopic disease; including "linkage disequilibrium" which occurs between a marker polymorphism (i.e., a DNA
polymorphism which is 'silent') and a functional polymorphism (i.e., genetic variation which affects phenotype or which contributes to a genetically determined trait) if the marker is situated in close proximity to the functional polymorphism (i.e., polyguanine, polycytosine) (p. 4, lines 1-10). Specifically, Hovnanian teaches the region in which a potential polyadenylation signal (AATAAA) is identifiable (SEQ ID NO. 3).
Regarding claims 18, 20, and 22-23, Hovnanian teaches an invention providing a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINK5 gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and screening for expression of the reporter gene product (p. 27, lines 1-10). Specifically, Hovnanian teaches that the above method of the invention can be used to identify compounds which up-regulate the expression of SPINK5 and hence have potential pharmacological activity in the treatment of Netherton's disease or atopic disease, including a promoter region or spacer of the human SPINK5 gene (i.e., sequences immediately upstream of the LEKTI transcription start site which are necessary for correctly positioning RNA polymerase and also to the proximal promoter region plus any additional sequence elements which may be involved in regulating LEKTI gene expression (p. 27, lines 20-30). Specifically, Hovnanian teaches that the LEKTI fusion proteins will typically be made by recombinant nucleic acid techniques in which two or more open reading frames that are translationally fused or may be chemically synthesized for purification and thus creating a shift in the reading frame (p. 21, lines 30-40).
Hovnanian also teaches that the previously described method can be used to target specific regions for mutational analyses, including sites of mononucleotide repeats (Example 2; p. 33, lines 25-30). Hovnanian specifically teaches that a plasmid vector comprising a reporter gene expression construct consisting of the LEKTI promoter region ligated to a
Promoter-less reporter gene cDNA and also a gene encoding a dominant selectable marker, such as neomycin phosphotransferase, is first constructed using standard molecular biology techniques (p. 29, lines 1-10).
Further, Hovnanian teaches that the previously described method of analysis via a nucleotide construct includes mutational analyses targeted based on mutations/variants occurring in an intron as a certain number of nucleotides + or – the nucleotide at the nearest exon/intron boundary (p. 18, lines 1-10).
Further, Hovnanian teaches that the previously described construct includes all or a part of the 5' and/or 3' untranslated regions of the LEKTI cDNA where the 5' untranslated region extends a further 43 nucleotides upstream of the initiating ATG codon and the 3' untranslated region extends a further 289 nucleotides downstream of the stop codon (p. 13, lines 20-30). Specifically, Hovnanian teaches the region in which a potential polyadenylation signal (AATAAA) is identifiable (SEQ ID NO. 3).
Regarding claim 29, Hovnanian teaches that the previously described method of analysis via a nucleotide construct includes mutational analyses targeted based on mutations/variants occurring in an intron as a certain number of nucleotides + or – the nucleotide at the nearest exon/intron boundary (p. 18, lines 1-10). Further, Hovnanian teaches that these specified introns can range between 5-50 bp or contain individualized guanines or cytosines (Table 6), as well as specified mutational patterns or transitions within SPINK5 (i.e., G22) (Table 1a; p. 17, lines 1-10).
Regarding claim 51-54, Hovnanian teaches a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINKS gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and screening for expression of the reporter gene product (p. 76, lines 30-40). Further, Hovnanian teaches that the eukaryotic host cell must be one which contains the appropriate transcription machinery for RNA Polymerase II transcription, and is preferably a cultured mammalian cell (p. 28, lines 25-30).
Further, Hovnanian teaches Stable integration into mammalian chromosomes may also be achieved by homologous recombination, a technique which has been commonly used to achieve
stable integration of foreign DNA into embryonic stem cells as a first stage in the construction of
transgenic mammals where stable integration into eukaryotic chromosomes can also be achieved by infection of a host cell with a retroviral vector (i.e., Cre, Flp, FIpO, Vika, and Dre) containing the appropriate reporter gene expression construct (p. 29, lines 30-40) and can be used to create physical maps of the host cell’s gene mappings (p. 31, lines 1-5). Further, Hovnanian teaches that the construct can be used to investigate targeted gene’s applications into cellular behaviors, including the expression of the SPINK5 gene within the thymus may indicate a role in T-cell maturation, or with antigen handling within other thymic cells (p. 48, lines 1-5).
Hovnanian teaches that the previously described construct in cells can be applied to genomic DNA that was extracted from peripheral blood following standard techniques after informed consent was obtained (p. 37, lines 20-25).
Hovnanian teaches each and every limitation of claims 1-11, 14, 17-18, 20, 22-23, 29, and 51-54, and therefore Hovnanian anticipates claim 1-11, 14, 17-18, 20, 22-23, 29, and 51-54.
Applicant’s Response: The Applicant argues that the 102 rejections, as shown above, is improper because Hovnanian does not teach the claimed nucleic acid construct as a whole, but merely mentions individual elements such as ORFs, mononucleotide repeats, and frameshift mutations in isolation. The Applicant contents that the Office improperly relied on keyword overlap rather than the specific structural arrangement required by the claims, particularly the positioning of the mononucleotide repeat and ORF out of frame with the translation start site. The Applicant further asserts that Hovnanian does not provide a single example that expressly discloses all claimed limitations in the required combination.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
As of note, anticipation does not require that the prior art disclose the claimed invention in ipissimis verbis or provide a single working example expressly mapping each limitation; rather, it is sufficient that the reference disclose or teach, either expressly or inherently, each and every element of the claim arranged as required. See MPEP 2131; In re Gleave, 560 F. 3d 1331, 1334 (Fed. Cir. 2009).
Notably, Hovnanian teaches nucleic acid constructs comprising promoter regions operably linked to report genes, open reading frames, mononucleotide repeat regions, and genetic alterations associated with SPINK5, including frameshift mutations, insertions, deletions, and polymorphic repeat regions (Example 2; p. 4, lines 1-10; p. 18, lines 1-10; p. 21, lines 30-40; p. 27, lines 20-30; p. 33, lines 25-30). Further, Hovnanian teaches fusions of open reading frames, reporter-based constructs and targeting specific genomic regions including sites of mononucleotide repeats for mutational analysis (p. 21, lines 30-40; p. 29, lines 10-15; p. 43, lines 35-40). The fact that the Applicant’s claims recite these elements in combination does not confer novelty where the same elements and their functional relationships are already disclosed in the reference. See MPEP 2131.01; In re Petering, 301 F.2d 676, 681 (CCPA 1962).
Further, to address the Applicant’s assertion that the Office has merely “searched for words: rather than meaning is not supported by the record. The Office Action (8/11/2025) identifies specific disclosures in Hovnanian that correspond to the claimed construct architecture, including operable linkage, ORFs, reporter constructs, and mononucleotide repeat-associated sequence variation. Anticipation does not require that Hovnanian describe the Applicant’s precise experimental purpose or preferred embodiment, only that the claimed structural and functional limitations are disclosed. See MPEP 2131; Titanium Metals Corp. v. Banner, 778 F. 2d 775, 782 (Fed. Cir. 1985).
The Applicant further argues that Hovnanian fails to teach a construct in which the mononucleotide repeats and ORF are “out of frame” relative to the translation start site. However, Hovnanian teaches frameshift mutations, fusion ORFs, and alterations to reading frames arising from insertions, deletions, and repeat-associated variation (Example 2; p. 4, lines 1-10; p. 18, lines 1-10; p. 21, lines 30-40; p. 27, lines 20-30; p. 33, lines 25-30), which inherently result in ORFs being out of frame with respect to the start codon. Where the natural and inevitable result of practicing the prior art includes the claimed arrangement, the limitation is inherently disclosed. See MPEP 2112; In re Cruciferous Sprout Litig., 301 F. 3d 1343, 1349 (Fed. Cir. 2002).
Finally, the Applicant argues that the Office has not pointed to a single explicit example in Hovnanian meeting all limitations is not sufficient to overcome anticipation. A reference anticipates if it discloses a genus that reasonably encompasses the claimed species. See MPEP 2131.02; In re Schaumann, 572 F. 2d 312, 316 (CCPA 1978). Hovnanian’s disclosure of SPINK5-related constructs, mononucleotide repeat variation, reporter systems, and altered ORFs reasonably encompasses the claimed subject matter.
Accordingly, because Hovnanian teaches each and every limitation of claims 1-11, 14, 17-18, 20, 22-23, 29, and 51-54, either expressly or inherently, the rejection under 35 USC 102 is maintained.
Claim Rejections - 35 USC § 103
Claims 12-13 are rejected under 35 U.S.C. 103 as being unpatentable over Hovnanian et al., (WO 0164747 A1, published 9/7/2001), as applied to claims 1-11, 14, 17-18, 20, 22-23, 29, and 51-54 above, in view of Viswanathan et al. (“High-performance probes for light and electron microscopy, Nat. Methods, published 12/1/2015).
As described above, Hovnanian teaches an invention providing a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINK5 gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and
screening for expression of the reporter gene product (p. 27, lines 1-10). Specifically, Hovnanian teaches that the above method of the invention can be used to identify compounds which up-regulate the expression of SPINK5 and hence have potential pharmacological activity in the treatment of Netherton's disease or atopic disease, including a promoter region or spacer of the human SPINK5 gene (i.e., sequences immediately upstream of the LEKTI transcription start site which are necessary for correctly positioning RNA polymerase and also to the proximal promoter region plus any additional sequence elements which may be involved in regulating LEKTI gene expression (p. 27, lines 20-30). Specifically, Hovnanian teaches that the LEKTI fusion proteins will typically be made by recombinant nucleic acid techniques in which two or
more open reading frames that are translationally fused or may be chemically synthesized
for purification and thus creating a shift in the reading frame (p. 21, lines 30-40).
Hovnanian does not teach or suggest the ORF encodes a tandem fusion of two or more spaghetti monster immunoreporter comprising 20 or more V5 epitope tags.
Viswanathan teaches an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins, where these molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location and these “spaghetti monster” fluorescent proteins (smFPs) distribute well in neurons, notably into small dendrites, spines and axons (Abstract). Further, Viswanathan teaches that almost all epitope tagging experiments draw upon a small set of validated peptide antigens, including influenza hemagglutinin, myelocytomatosis viral oncogene (myc), simian virus 5-derived epitope (V5), the synthetic peptide FLAG, the synthetic streptavidin-binding strep-tag, and more recently OLLAS (Escherichia coli OmpF linker and mouse langerin) and Sun Tag, and specifically, the small size of epitope tags (typically 8–12 amino acids) enables their attachment to gene targets (Introduction: Paragraph 1). Viswanathan also teaches antigenic protein tags called “spaghetti monster” fluorescent proteins (smFPs), which contain approximately 15 copies of single epitope tags strategically inserted into an FP scaffold with either an intact or darkened chromophore (Introduction: Paragraph 4).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Hovnanian’s teaching of immunoreceptor fusion constructs with Viswanathan’s specific “spaghetti monster” multi epitope tag technology to create improved detection and visualization systems. Both references address the same field of genetic report systems and seek to enhance protein detection and visualization. Further, one skilled in the art would have a reasonable expectation of success in combining these teachings because both references demonstrate functional multi-epitope tag systems, and Viswanathan specifically shows that approximately 15 epitope tags can be successfully incorporated while maintaining protein function. Under MPEP 2144.05, where Viswanathan teaches approximately 15 copies and the claims specify “20 or more V5 epitope tags”, the ranges overlap and a person of ordinary skill in the art would recognize that increasing from 15 to 20+ epitope tags represent routine optimization within the established parameters.
Applicant’s Response: The Applicant argues that the cited references fail to teach or suggest the claimed combination for sparse and stochastic cellular labeling, asserting that Hovnanian is directed to disease gene identification and screening rather than intentional sparse labeling to visualize complete cell morphology. The Applicant further contends that Viswanathan (and the other secondary references) are concerned with enhancing signal intensity or epitope tagging, not regulating the fraction of labeled cells, and therefore do not supply a motivation to combine with Hovnanian. And, thus, the Applicant argues that the Office has not established a proper rationale or reasonable expectation of success for modifying Hovnanian to achieve the claimed sparse, percentage-based labeling system.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
As discussed above, Hovnanian teaches nucleic acid constructs comprising promoter regions operably linked to report genes, open reading frames, mononucleotide repeat regions, and genetic alterations associated with SPINK5, including frameshift mutations, insertions, deletions, and polymorphic repeat regions (Example 2; p. 4, lines 1-10; p. 18, lines 1-10; p. 21, lines 30-40; p. 27, lines 20-30; p. 33, lines 25-30). Further, Hovnanian teaches fusions of open reading frames, reporter-based constructs and targeting specific genomic regions including sites of mononucleotide repeats for mutational analysis (p. 21, lines 30-40; p. 29, lines 10-15; p. 43, lines 35-40). Viswanathan teaches an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins, where these molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location and these “spaghetti monster” fluorescent proteins (smFPs) distribute well in neurons, notably into small dendrites, spines and axons (Abstract), including V5, and further demonstrates that incorporation of approximately 15 epitope tags is functional and suitable for enhanced detection and visualization (Introduction: Paragraphs 1-4).
The Applicant argues that neither reference teaches a tandem fusion comprising 20 or more V5 epitope tags and that increasing the number of tags would not be obvious. However, Viswanathan expressly teaches multi-epitope tag systems and establishes that increasing the number of epitope tags is a known technique for enhancing detection sensitivity. Modifying the fusion constructs of Hovnanian to incorporate a greater number of epitope tags as taught by Viswanathan would have been an obvious design choice to improve signal strength and visualization. The claimed range of “20 or more” epitope tags reasonably overlaps with the approximate 15-tag constructs taught by Viswanathan, and increasing the number of tags represents routine optimization of a known result-effective variable. See MPEP 2144.05; In re Peterson, 315 F. 3d 1325, 1330 (Fed. Cir. 2003).
The Applicant further asserts that the references have different objectives and that no motivation to combine has been established. This argument is not persuasive because both Hovnanian and Viswanathan are directed to genetic reporter systems and protein detection/visualization, and both seek to enhance detectability of expressed constructs. A person of ordinary skill in the art would have been motivated to combine Hovnanian’s fusion constructs with Viswanathan’s multi-epitope tagging strategy to improve detection and visualization, with a reasonable expectation of success given Viswanathan’s demonstration of functional multi-epitope tags. See MPEP 2131 and 2143; KSR Int’l Co. v. Teleflex Inc., 550 US 398, 417 (2007).
Accordingly, claims 12-13 would have been obvious over Hovnanian in view of Viswanathan, and the rejection under 35 USC 103 is maintained.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Hovnanian et al., (WO 0164747 A1, published 9/7/2001), as applied to claims 1-11, 14, 17-18, 20, 22-23, 29, and 51-54 above, in view of Ahearn et al. (“Regulating the regulator: post-translational modification of RAS”, Nature Reviews, published 12/22/2011).
As described above, Hovnanian teaches an invention providing a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINK5 gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and
screening for expression of the reporter gene product (p. 27, lines 1-10). Specifically, Hovnanian teaches that the above method of the invention can be used to identify compounds which up-regulate the expression of SPINK5 and hence have potential pharmacological activity in the treatment of Netherton's disease or atopic disease, including a promoter region or spacer of the human SPINK5 gene (i.e., sequences immediately upstream of the LEKTI transcription start site which are necessary for correctly positioning RNA polymerase and also to the proximal promoter region plus any additional sequence elements which may be involved in regulating LEKTI gene expression (p. 27, lines 20-30). Specifically, Hovnanian teaches that the LEKTI fusion proteins will typically be made by recombinant nucleic acid techniques in which two or
more open reading frames that are translationally fused or may be chemically synthesized
for purification and thus creating a shift in the reading frame (p. 21, lines 30-40).
Hovnanian does not teach or suggest the ORF is fused with a farnesylation signal that is a Ras CAAX domain.
Ahearn teaches RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes (Abstract). Specifically, Ahearn teaches that important RAS proteins include the constitutive and irreversible remodeling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerization, monoubiquitylation, deubiquitylation, nitrosylation, ADP ribosylation and glycosylation (Abstract). Further, Ahearn teaches that cell biologists have also devoted attention to RAS because it represents the archetypal CAAX protein, where this class of protein terminates in a CAAX sequence, in which C is Cys, A is usually, but not always, an aliphatic amino acid and X is any amino acid and the CAAX sequence directs the post-translational modification (PTM) of the carboxyl terminus of the protein with a polyisoprenoid lipid which, in the case of RAS, is a farnesyl moiety (Main: Paragraphs 1-2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Hovnanian’s teaching of immunoreceptor fusion constructs with Ahearn’s well-known RAS CAAX farnesylation signals to enhance protein localization and membrane association. Both references address protein modification systems and farnesylation signals are commonly used in the field to direct proteins to cellular membranes for improved functionality. Further, one skilled in the art would have a reasonable expectation of success because Ahearn demonstrates that CAAX domains effectively direct farnesylation, and such modifications are routinely added to fusion proteins to enhance their cellular localization and biological activity. The combination represents standard practice in protein engineering.
Applicant’s Response: The Applicant argues that the cited references fail to teach or suggest the claimed combination for sparse and stochastic cellular labeling, asserting that Hovnanian is directed to disease gene identification and screening rather than intentional sparse labeling to visualize complete cell morphology. The Applicant further contends that Viswanathan (and the other secondary references) are concerned with enhancing signal intensity or epitope tagging, not regulating the fraction of labeled cells, and therefore do not supply a motivation to combine with Hovnanian. And, thus, the Applicant argues that the Office has not established a proper rationale or reasonable expectation of success for modifying Hovnanian to achieve the claimed sparse, percentage-based labeling system.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
As discussed above, Hovnanian teaches nucleic acid constructs comprising promoter regions operably linked to report genes, open reading frames, mononucleotide repeat regions, and genetic alterations associated with SPINK5, including frameshift mutations, insertions, deletions, and polymorphic repeat regions (Example 2; p. 4, lines 1-10; p. 18, lines 1-10; p. 21, lines 30-40; p. 27, lines 20-30; p. 33, lines 25-30). Further, Hovnanian teaches fusions of open reading frames, reporter-based constructs and targeting specific genomic regions including sites of mononucleotide repeats for mutational analysis (p. 21, lines 30-40; p. 29, lines 10-15; p. 43, lines 35-40). Ahearn teaches RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes (Abstract). Specifically, Ahearn teaches that important RAS proteins include the constitutive and irreversible remodeling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerization, monoubiquitylation, deubiquitylation, nitrosylation, ADP ribosylation and glycosylation (Abstract). And, thus, since Ahearn establishes that CAAX farnesylation signals are well-known and commonly appended to proteins of interest to enhance membrane targeting and biological activity, Ahearn teaches that fusion of a CAAX domain to a protein is a predictable and established modification to improve localization and functional performance.
Although the Applicant argues that the cited references do not suggest modifying Hovnanian’s constructs to include such post-translational modification signals and that the Office has failed to establish a motivation to combine, this argument is not persuasive because Ahearn expressly teaches that incorporation of farnesylation and related motifs is a routine and predictable technique used by those of ordinary skill in the art to control protein behavior and localization. Applying these known regulatory signals to Hovnanian’s expressed fusion proteins would have been an obvious design choice to modulate cellular localization or functional output of the reporter constructs, with a reasonable expectation of success. See MPEP 2141 and 2143l KSR Int’l Co. v Teleflex Inc., 550 US 398, 417 (2007).
Further, the addition of a well-known post-translational modification signal to an otherwise known construct represents no more than the predictable use of prior art elements according to their established functions. See MPEP 2144; In re Keller, 642 F. 2d 413, 425 (CCPA 1981). The Applicant has not provided evidence of unexpected results or technical incompatibility that would overcome the prima facie case of obviousness.
Accordingly, claims 15-16 would have been obvious over Hovnanian in view of Ahearn, and the rejection under 35 USC 103 is properly maintained.
Claims 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Hovnanian et al., (WO 0164747 A1, published 9/7/2001), as applied to claims 1-11, 14, 17-18, 20, 22-23, 29, and 51-54 above, in view of Madisen et al. (“A robust and high-throughput Cre reporting and characterization system for the whole mouse brain”, Nat. Neurosci., published 7/1/2010).
As described above, Hovnanian teaches an invention providing a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINK5 gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and
screening for expression of the reporter gene product (p. 27, lines 1-10). Specifically, Hovnanian teaches that the above method of the invention can be used to identify compounds which up-regulate the expression of SPINK5 and hence have potential pharmacological activity in the treatment of Netherton's disease or atopic disease, including a promoter region or spacer of the human SPINK5 gene (i.e., sequences immediately upstream of the LEKTI transcription start site which are necessary for correctly positioning RNA polymerase and also to the proximal promoter region plus any additional sequence elements which may be involved in regulating LEKTI gene expression (p. 27, lines 20-30). Specifically, Hovnanian teaches that the LEKTI fusion proteins will typically be made by recombinant nucleic acid techniques in which two or
more open reading frames that are translationally fused or may be chemically synthesized
for purification and thus creating a shift in the reading frame (p. 21, lines 30-40).
Hovnanian does not teach or suggest that the recombinase binding sites are LoxP sites, and wherein the LoxP sites are oriented such that Cre recombinase excises the transcriptional stop sequence; or that the recombinase binding sites are Frt sites, and wherein the Frt sites are oriented such that Flp recombinase excises the transcriptional stop sequence; or that the promoter is a cytomegalovirus early enhancer element and chicken beta actin (CAG) promoter.
Madisen teaches that the Cre/lox system is widely used in mice to achieve cell-type-specific gene expression, however, a strong and universal responding system to express genes under Cre control is still lacking (Abstract). Further, Madisen teaches that in addition to the traditional Cre/lox pairing, a pair of FRT sites was incorporated to allow for a Flp recombinase-mediated replacement strategy (“Flp-in”) to swap other genes into the same locus at high efficiency (>90%) using a double-selection strategy (Results: Paragraph 1). Specifically, Madisen teaches that in order to target specified neurons, in utero electroporation or recombinant viruses (i.e., woodchuck hepatitis virus), robust expression has been difficult to obtain in transgenic mice (Introduction: Paragraph 2). Madisen also teaches further modifications by targeted insertion of a construct containing the strong and ubiquitous CAG promoter, followed by a floxed-Stop cassette-controlled fluorescent marker gene (Results: Paragraphs 1-2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Hovnanian’s teaching of immunoreceptor fusion constructs with Madisen’s well-established Cre/lox and Flp/FRT recombinase systems and CAG promoter to achieve robust, cell-type-specific gene expression with controllable activation. One skilled in the art would have a reasonable expectation of success because Madisen demonstrates that these recombinase systems and CAG promoter work effectively for controlled gene expression, further expounding on the interchangeability of virus types (i.e., cytomegalovirus) for gene delivery dependent on the specific targeting requirements. As such, these systems are standard tools in the field for creating conditional expression constructs.
Applicant’s Response: The Applicant argues that the cited references fail to teach or suggest the claimed combination for sparse and stochastic cellular labeling, asserting that Hovnanian is directed to disease gene identification and screening rather than intentional sparse labeling to visualize complete cell morphology. The Applicant further contends that Viswanathan (and the other secondary references) are concerned with enhancing signal intensity or epitope tagging, not regulating the fraction of labeled cells, and therefore do not supply a motivation to combine with Hovnanian. And, thus, the Applicant argues that the Office has not established a proper rationale or reasonable expectation of success for modifying Hovnanian to achieve the claimed sparse, percentage-based labeling system.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
As discussed above, Hovnanian teaches nucleic acid constructs comprising promoter regions operably linked to report genes, open reading frames, mononucleotide repeat regions, and genetic alterations associated with SPINK5, including frameshift mutations, insertions, deletions, and polymorphic repeat regions (Example 2; p. 4, lines 1-10; p. 18, lines 1-10; p. 21, lines 30-40; p. 27, lines 20-30; p. 33, lines 25-30). Further, Hovnanian teaches fusions of open reading frames, reporter-based constructs and targeting specific genomic regions including sites of mononucleotide repeats for mutational analysis (p. 21, lines 30-40; p. 29, lines 10-15; p. 43, lines 35-40). Although Hovnanian does not teach Cre/lox or Flp/FRT recombinase control or a CAG promoter, Madisen teaches robust and high-throughput gene expression systems in mice employing Cre/loxP and Flp/FRT recombinase strategies in combination with a strong, ubiquitous CAG promoter and a floxed transcriptional stop cassette to achieve controllable, cell-type-specific gene expression (Abstract; Results: Paragraphs 1-2).
Although the Applicant argues that the cited references fail to teach or suggest the claimed combination for sparse and stochastic cellular labeling and that Hovnanian is directed to disease gene screening rather than morphology visualization, this argument is not persuasive because the claims are not limited to a specific intent or end use, and obviousness does not require the references to share the same stated objective. See MPEP 2141 and KSR Int’l Co. v. Teleflex Inc., 550 US 398, 417 (2007). Madisen teaches recombinase-controlled activation of reporter constructs as a standard and effective approach for controlled gene expression, and applying these well-established recombinase systems and promoters to Hovnanian’s reporter constructs would have been an obvious modification to achieve regulated activation of expression.
The Applicant further contends that the secondary references do not provide a motivation to combine or a reasonable expectation of success. This argument is not persuasive because Madisen demonstrates that Cre/loxP and Flp/FRT systems, in combination with CAG promoter, reliably provide robust, conditional gene expression in transgenic systems. A person of ordinary skill in the art would have been motivated to incorporate these known regulatory elements into Hovnanian’s constructs to obtain controllable activation of reporter expression, with a reasonable expectation of success given their widespread and predictable use. See MPEP 2143 and 2144; In re Keller, 642 F. 2d 413, 425 (CCPA 1981).
Accordingly, claims 24-26 would have been obvious over Hovnanian in view of Madisen, as the claimed subject matter represents the predictable use of prior art elements according to their established functions. The rejection of claims 24-26 under 35 USC 103 is therefore maintained.
New Rejections
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.
Claims 55-60 are rejected under 35 U.S.C. 103 as being unpatentable over Hovnanian et al., (WO 0164747 A1, published 9/7/2001), as applied to claims 1-11, 14, 17-18, 20, 22-23, 29, and 51-54 above, in view of Viswanathan et al. (“High-performance probes for light and electron microscopy, Nat. Methods, published 12/1/2015).
As described above, Hovnanian teaches an invention providing a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINK5 gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and
screening for expression of the reporter gene product (p. 27, lines 1-10). Specifically, Hovnanian teaches that the above method of the invention can be used to identify compounds which up-regulate the expression of SPINK5 and hence have potential pharmacological activity in the treatment of Netherton's disease or atopic disease, including a promoter region or spacer of the human SPINK5 gene (i.e., sequences immediately upstream of the LEKTI transcription start site which are necessary for correctly positioning RNA polymerase and also to the proximal promoter region plus any additional sequence elements which may be involved in regulating LEKTI gene expression (p. 27, lines 20-30). Specifically, Hovnanian teaches that the LEKTI fusion proteins will typically be made by recombinant nucleic acid techniques in which two or
more open reading frames that are translationally fused or may be chemically synthesized
for purification and thus creating a shift in the reading frame (p. 21, lines 30-40).
Hovnanian teaches a method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises: providing a recombinant host cell containing a reporter gene expression construct comprising the promoter region of the human SPINKS gene operably linked to a reporter gene; contacting the host cell with a candidate compound; and screening for expression of the reporter gene product (p. 76, lines 30-40). Further, Hovnanian teaches that the eukaryotic host cell must be one which contains the appropriate transcription machinery for RNA Polymerase II transcription, and is preferably a cultured mammalian cell (p. 28, lines 25-30).
Further, Hovnanian teaches stable integration into mammalian chromosomes may also be achieved by homologous recombination, a technique which has been commonly used to achieve
stable integration of foreign DNA into embryonic stem cells as a first stage in the construction of
transgenic mammals where stable integration into eukaryotic chromosomes can also be achieved by infection of a host cell with a retroviral vector (i.e., Cre, Flp, FIpO, Vika, and Dre) containing the appropriate reporter gene expression construct (p. 29, lines 30-40) and can be used to create physical maps of the host cell’s gene mappings (p. 31, lines 1-5). Further, Hovnanian teaches that the construct can be used to investigate targeted gene’s applications into cellular behaviors, including the expression of the SPINK5 gene within the thymus may indicate a role in T-cell maturation, or with antigen handling within other thymic cells (p. 48, lines 1-5).
Specifically, Hovnanian teaches that polymorphisms identified within the Netherton’s gene encompasses a frequency most commonly targeting 3.6% (Table 2).
Hovnanian teaches that the previously described construct in cells can be applied to genomic DNA that was extracted from peripheral blood following standard techniques after informed consent was obtained (p. 37, lines 20-25).
Hovnanian does not teach or suggest targeted and precise stochastic labeling to express one or more-site specific recombinase.
Viswanathan teaches an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins, where these molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location and these “spaghetti monster” fluorescent proteins (smFPs) distribute well in neurons, notably into small dendrites, spines and axons (Abstract). Further, Viswanathan teaches improved labeling of cells and single molecule tracking efficiency of proteins in fixed and live preparations; including sparsely labeled cells, from apical dendrites through soma to distal dendrites (Figure 3a-3d). Viswanathan also teaches high single-molecule tracking photon counts from orthogonal tags and antibodies facilitate robust simultaneous multi-color molecule tracking in cells and in vivo, where the high avidity of the smFP-antibody interactions increase signal-to-noise in RNA-Seq and ChIP-Seq experiments targeting sparse cell types, increasing reliability of brain transcriptomics (Discussion: Paragraphs 1-3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the reporter constructs and fusion proteins taught by Hovnanian to incorporate the multi-epitope tagging strategies taught by Viswanathan in order to improve detection sensitivity, visualization, and analytical robustness of SPINK5-associated reporter expression. Both references are directed to genetic reporter systems and protein detection in mammalian cells, and both seek to enhance the ability to detect and analyze expressed gene products. Therefore, the combination represents the predictable use of prior art elements according to their established functions. Further, a person of ordinary skill in the art would have had a reasonable expectation of success in combining these teachings because Viswanathan demonstrates that multi-epitope fluorescent tags function effectively when fused to target proteins in mammalian cells within the frequency range expressed in the instant claims, including under conditions of sparse labeling, and Hovnanian already teaches compatible mammalian expression systems and fusion-based reporter constructs. Therefore, the claimed subject matter does not require any modification beyond routine optimization of known reporter and tagging technologies.
Conclusions
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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 extension fee 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 date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached on 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684