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 on 2/2/2024. Claims 1-17 are pending. All pending claims are currently under examination.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO’s electronic filing system (see Section I.1 of the Legal Framework for EFS-Web or Patent Center (https://www.uspto.gov/patents-application- process/filing-online/legal-framework-efs-web), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via EFS-Web or Patent Center as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via EFS-Web or Patent Center as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - The Incorporation by Reference paragraph required by 37 CFR 1.821(c)(1) is missing or incomplete. See item 1) a) or 1) b) above. In particular, the sequence incorporation statement in the specification refers to the sequence listing file in terms of “kilobytes,” but must refer to the size of the file in terms of “bytes.” See MPEP 2422.03, section I, “ASCII Text File Submitted VIA EFS-Web.”
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 8-10, 11-13, and 16-17 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 8, claim 8 recites “the promoter of a target gene is demethylated through the use of a gene targeting guide RNA and a polynucleotide encoding a dCas9-TET1CD fusion peptide are co-transfected with into tissues of a diabetic patient.” Claim 8 is in general unclear because the subject of the sentence changes partway through the claim which creates subject-verb disagreement between “the promoter” and “are co-transfected.” Claim 8 further recites “co-transfected with into tissues of a diabetic,” which is unclear language. Claim 8 should be amended to recite “the promoter of a target gene is demethylated through the use of a gene targeting guide RNA and a polynucleotide encoding a dCas9-TET1CD fusion peptide that are co-transfected into tissues of a diabetic patient” to clarify the claim.
Claims 9-10 depend from claim 8 and do not resolve this 112(b) issue and are therefore also rejected.
Regarding claim 11, claim 11 recites the phrase “an improved method” (line 1). Recitation of the phrase “an improved method” is unclear because it is unclear as to what metric the method is being compared to establish an improvement (i.e., improved as compared to what?).
Claims 12-13 depend from claim 11 and do not resolve this 112(b) issue and are therefore also rejected.
Regarding claim 16, claim 16 recites “the targeted gene is TP53.” Claim 16 depends from claim 14, which recites “cocktail that targets demethylation of genes.” Hence, claim 14 recites that multiple genes are targeted in the cocktail. Claim 16 is confusing because by reciting “the gene” it becomes unclear how many genes are meant to be targeted in the method, whether the method is required to target multiple genes in the cocktail (as recited in claim 14) or just one gene (claim 16). Furthermore, claim 16 is confusing because the claim from which it depends, claim 14, recites multiple “genes.” Recitation of “the gene” in claim 16 therefore lacks clear antecedent basis, as it is unclear as to which of the multiple genes recited in claim 14 are being recited in claim 16.
Claim 17 depends from claim 16 and does not address this 112(b) issue and is therefore also rejected.
Claim Rejections - 35 USC § 112 - Enablement
Claims 1-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for: reducing diabetes-induced epigenetic barriers in mice where the epigenetic modulator is 5’-azacytidine (claims 1-5) or a dCas9-TET1CD with gRNA targeting TP53 in mice (claims 6-10) and a method to normalize high blood glucose levels mice using a combination of 5’azacytidine in combination with PDX1, FGF21, GLP-1R, and MafA (amino acids with 100% identity to SEQ ID NOs 2,4,6,8, claims 11-13), or a method of enhancing wound repair in keratinocytes by introducing demethylation agents consisting of a dCas9-TET1CD with gRNA targeting TP53 (claims 14-17), does not reasonably provide enablement for the scope of the genus “epigenetic modulator” (claims 1-13) or “demethylation cocktail” (claims 14-17) nor their administration or use in a method in cells or treatments other than in laboratory mice (claims 1-17), nor is the application enabling for mutations in SEQ ID NOs 2,4,6,8 as recited in claims 11-13. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 U.S.C. 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988). Wands states, on page 1404:
Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of these in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
Nature of the Invention/Scope of the Claims
Regarding independent claim 1, claim 1 is broadly drawn to a method of reducing diabetes-induced barriers in cells by introducing an epigenetic modulator into the cells. The fact that claim 1 recites “diabetes-induced” implicates that the cells are within a diabetic subject (i..e, “diabetes-induced barriers” in cells must be induced by a diabetic condition within an organism). Claim 1 therefore broadly encompasses the genus of “epigenetic modulator” to be administered into any cell type with the effect of reducing epigenetic barriers associated with diabetes, including into subjects such as humans. For instance, dependent claim 2 recites that the reduction in epigenetic barriers is used to treat a diabetic condition such as hyperglycemia, which reasonably includes in a human diabetic patient. This claim language is problematic because the Applicant has not identified a structural-functional relationship between the term “epigenetic modulator” and its effect of reducing epigenetic barriers. As such, the molecule “epigenetic modulator” is undefined and could potentially be any number of molecules or compositions. For instance, the “epigenetic modulator” is recited as an inhibitor of methyltransferase in claim 4, such as 5’-azacytidine (claim 5), and is also recited to be a dead Cas9 nuclease fusion protein which targets specific genes for demethylation (claims 6-8). Thus, the term “epigenetic modulator” is any potential molecule, Cas nuclease, or guide RNA to a specific gene. Furthermore, as will be discussed below, even defined epigenetic modulators such as 5’-azacytidine are known to have unpredictable effects in different cell types and organism which were not taught or envisioned by the Applicant.
Claims 6-10 recite that the epigenetic modulator is targeted to specific genes. This claim language is problematic because diabetes (“diabetes-induced epigenetic barriers”) is known to be a complex disease involving multiple genes which are not all characterized in the art (see below). Thus, the specification is not enabling for the broad genus of “specific genes” because such genes are not well-understood in the context of diabetes (see below).
Claims 11-13 recite a genus of amino acid sequences that are 95% identical to SEQ ID NOs 2,4,6 and 8. This claim language is problematic because the scope of the claim includes mutations to SEQ ID NOs 2,4,6, and 8, where the art teaches that mutations in such proteins can have profound and unpredictable effects on said proteins, where the Applicant has not characterized or provided guidance with respect to which residues could be mutated to retain functionality (see below). Furthermore, claim 11 recites “normalizing blood glucose” however the specification does not enable a method for normalizing low blood glucose because the data only support lowering blood glucose, not raising it (see below).
Claims 14-17 recite a “demethylation cocktail” that targets the demethylation of genes. Claims 14-17, like claims 6-10, therefore recite a broad genus of target genes which are not fully characterized in the art (see below).
Guidance in the Specification
Regarding the guidance provided in the specification, the Applicant provides Examples 1-2 (see pages 14-16). In Example 1, the Applicant injects a cocktail of PDX1, MafA, GLP-1R, and FGF21 into a diabetic mouse model (page 14). The Applicant observed that the laboratory mice either responded to the cocktail or did not respond to the cocktail (“responder” and “non-responder”, page 14, final paragraph). To investigate the role of epigenetic modulation, the Applicants applied the epigenetic modulator 5’-azacytidine and saw that the non-responders had lower blood glucose levels (page 15, first paragraph). In Example 2, the Applicant has used a different epigenetic modulator, namely, a dCas9-TET1CD fusion system which targets specific genes for demethylation. In Example 2 (pages 15-16), the Applicant targeted the gene TP53 using the dCas9-TET1CD system, where wound healing was observed upon treatment with the modulator in mice.
As an initial matter, as discussed above, the Applicant has not identified a specific structure or molecule to define the term “epigenetic modulator,” and the claims are therefore reciting a molecule by its function without guidance in the specification regarding the structure or composition requirements of the “epigenetic modulator.”
Secondly, the Applicant does not demonstrate that the 5’-azacytidine modulator alone could be used in a method to treat any diabetic condition, as presently recited in claim 2, because 5’-azacytidine is used in conjunction with the PDX1, MafA, GLP-1R, and FDF21 cocktail in order to achieve any treatment effect.
Thirdly, the Applicant has only tested one target gene: TP53. The Applicant has not elucidated or given guidance to show that other genes could be targeted for demethylation to have any effect, or provided guidance with respect to what genes should be targeted using the dCas9-TET1CD gRNA systems recited.
Fourth, the Applicant is broadly reciting the term “epigenetic modulator.” However, the Applicant has only offered two brief examples, both of which focus on and target exclusively the epigenetic modification “methylation” (i.e., the dCas9-TET1CD system demethylates promoters where 5’-azacytidine is an inhibitor of methyltrasnsferase). However, the Applicant has not addressed any other forms of epigenetics, such as histone acetylation, which is also known to be associated with diabetic conditions (see discussion on state of the art, below).
Finally, the Applicant has tested a mouse model, and not shown that such methods could be applied to other organisms.
The Applicant has therefore offered one example using 5’-azacytidine in combination with a cocktail of PDX1, FGF21, GFP-1R, and MafA to show reduction in blood glucose levels (Example 1) and one example of a dCas9-TET1CD system targeting the gene TP53 for demethylation using a guide RNA to show improved wound healing (Example 2).
Furthermore, Example 1 shows variable responses in responder vs. non-responder mice with respect to the PDX1, GLP-1R, MafA, and FDF21 cocktail. As discussed further below, the pathophysiology and specific contributions of epigenetics in diabetes is unknown and uncharacterized. Given that the Applicant’s data shows variability even amongst a clonal population of laboratory mice, the complexity of the epigenetic landscape and its role in diabetes is further compounded by the fact that the disease is not fully characterized or understood at the epigenetic level (see below, discussion of Ponard). The Applicant’s variable data in the clonal population of mice speaks to the unpredictability and uncertainty of what specific epigenetic modifications are involved in diabetic manifestations, as no specific explanation for responder vs. non-responder with respect to the PDX1, GLP-1R, FDF21, and MafA cocktail is presented.
State of the Art
Regarding the state of the art, it is known that the introduction of 5’-azatytidine results in unpredictable results in diabetics. For instance, Ponard (Ponard A et al. J Med Case Rep. 2018 Jul 3;12(1):199) is a case study where 5’-azatytidine, the epigenetic modulator tested in Example 1 and presently claimed, was introduced into a diabetic patient (Title, Abstract, and throughout). Ponad teaches that the pathophysiology of diabetes is not well understood, where epigenetics are implicated in the disease (Abstract). Ponard therefore teaches that, in general , the physiological responses and pathology of diabetes and its epigenetic status are not fully characterized or understood (Abstract). Furthermore, Ponard teaches that upon administration of 5’-azacytidine (the epigenetic modulator presently recited), the diabetic patient in the case study saw in increase in blood glucose dysregulation, where blood glucose levels were increased requiring an increase in insulin dosage to counteract the effect (Abstract, “Case Study”). Ponard further teaches that when treating with a hypomethylation agent (i..e, an epigenetic modulator, and in particular 5’-azacytidine), doctors are advised to increase glucose monitoring owing to the unknown effects of such classes of drugs (Abstract, Conclusion). Thus, Ponard teaches that it is known in the art that the pathophysiology and contribution of epigenetics to diabetic cells/tissues/disease states is unknown and uncharacterized, where furthermore the introduction of epigenetic modulators such as 5’-azacytidine or other “hypomethylation agents” can in fact further exacerbate diabetic disease conditions and worsen hyperglycemia, where the mechanism of such dysregulation is not fully understood or characterized (see Abstract and Discussion). Thus, the Applicant has not shown enablement for the use of epigenetic modulators such as 5’-azacytidine to disrupt uncharacterized epigenetic barriers (claim 1), or to treat conditions such as hyperglycemia (claim 2), or to normalize blood glucose levels (claims 11-13) owing to the unpredictable effects of introducing such epigenetic modulators into cells, where it is known that such introduction leads to hyperglycemia and exacerbates diabetes in human subjects (Ponard, Abstract and Discussion). Thus, the disclosure in the specification of mouse model data related to the introduction of 5’azacytidine does not apply to humans, as taught by Ponard, who teaches that the introduction of 5’azacytidine has unpredictable effects in human diabetics which are different from those observed and contemplated by the Applicant (see above). The Applicant is therefore enabled only for the administration of such compounds in laboratory conditions/mice which they have tested, which do not appear to apply to other scenarios such as human cells, tissues, and patients. Furthermore, Ponard teaches that such unpredictability applies to the broad class of “hypomethylation agents” which includes all of the epigenetic modulators presently recited including the dCas9-TET1CD gRNA system.
For a point of clarification, Ponard teaches “5’azacitidine” which, as evidenced by Millipore (product information for 5’-azacytidine, published 2026) is a synonym for 5’azacytidine (see page 1 of Millipore).
Furthermore, the Applicant is broadly reciting “epigenetic” modulators, to include modulation of epigenetics changes including acetylation. It is known in the art that acetylation is an epigenetic modification that is associated with diabetes. For instance, Kumar (Kumar KK et al. Clin Epigenetics. 2024 Jun 11;16(1):78) teaches that:
“Epigenetic modifiers, histone deacetylases (HDACs), are enzymes that remove acetyl groups from histones and play an important role in a variety of molecular processes, including pancreatic cell destiny, insulin release, insulin production, insulin signaling, and glucose metabolism. HDACs also govern other regulatory processes related to diabetes, such as oxidative stress, inflammation, apoptosis, and fibrosis, revealed by network and functional analysis. This review explains the current understanding of the function of HDACs in diabetic pathophysiology, the inhibitory role of various HDAC inhibitors (HDACi), and their functional importance as biomarkers and possible therapeutic targets for T2DM. While their role in T2DM is still emerging, a better understanding of the role of HDACi may be relevant in improving insulin sensitivity, protecting β-cells and reducing T2DM-associated complications, among others.” (Abstract).
Thus, Kumar teaches that acetylation is an epigenetic modification that plays a complex and important role in diabetes, where acetylation is not only complex but is not fully characterized in its role in diabetes (Abstract). Given the unpredictable and uncharacterized role of acetylation in diabetes, and the lack of testing or guidance with respect to such epigenetic modifications or regulators which would target such modifications, the Applicant has a higher burden of demonstrating and reducing to practice such epigenetic modulators which would specifically target acetylation modifications. The Applicant has only addressed “methylation” as an epigenetic modification in their Examples.
Furthermore, with respect to specific “gene” targets such as using the dCas9-TET1CD system recited in claims 6-10 and 14-17, it is known in the art that identifying specific methylation patterns in genes to correlate to diabetes and/or their functional role or link to diabetes is complex and not well understood. For instance, Nadiger (Nadiger N et al. Clin Epigenetics. 2024 May 16;16(1):67) is a review article that focuses on identifying methylation patterns in diabetics (Title, Abstract, throughout). Nadiger performed comprehensive literature reviews of known methylation-associations in diabetes, and furthermore concludes that “Although the majority of the top differentially methylated genes are well known, other more recent genes reported here should be investigated further to understand their role in pathogenesis of T2DM,” (see Abstract and page 22, left column, third paragraph). Thus, Nadiger teaches that after filing of the application, new potential gene targets are still being discovered which are associated with DNA methylation and the onset of diabetes. Furthermore, Nadiger performed extensive literature review and searches and demonstrated that methylation patterns vary widely across tissue and cell types (Abstract, and throughout). Thus, the recited invention is complex in the sense that specific targeted methylation genes are in general unknown, and also change based upon cell and tissue target. Furthermore, Nadiger teaches that conditions such as obesity which often co-occur with diabetes is also dependent upon DNA methylation patterns (e.g., page 14, left column). Thus, methylation states in diabetics also comprise complex methylation patterns of numerous genes associated with other conditions such as obesity (page 14, left column).
Undue Experimentation
The Applicant is burdened with undue experimentation in order to practice the recited methods as claimed. For instance, the practitioner would be required to identify epigenetic modulators not recited and taught in the specification and test them in the physiologically complex and undefined disease state diabetes. The complexity and burden are further compounded by the fact that it is known in the art that such hypomethylation agents such as 5’-azacytidine such as that recited is known to exacerbate diabetic conditions such as hyperglycemia (per Ponard, see above). Thus, the practitioner is burdened with having to not only identify a potential drug class and molecule type but also to test such drugs in diabetic-specific conditions for effect, where the art teaches that such drugs are known to be counterproductive and harmful in diabetic conditions in humans (per Ponard). Furthermore, the Applicant would be experimentally burdened by having to test additional epigenetic modulators not tested in the specification such as acetylation modulators, where such epigenetic modifications such as acetylation is known to be associated with diabetes in a complex network (per Kumar). Furthermore, the Applicant would be burdened with having to determine which specific gRNA to use to target what specific genes in diabetic conditions, which is a complex task requiring the identification of methylated genes, which are still being discovered and have not all been characterized or discovered (per Nadiger).
Furthermore, regarding claim 16, while claim 16 recites that the gene is TP53, claim 16 depends from claim 14 which recites a cocktail of target genes that are demethylated. The Applicant has not identified target genes to be demethylated, where the scope of the category “cocktail of target genes” for wound repair is highly unpredictable and must be defined empirically, per Nadiger.
Additional 112(a) Enablement Issues for Claims 11-13
In addition, claims 11-13 are rejected for the following enablement issues:
Regarding claim 11, claim 11 is broadly drawn to a method of normalize blood glucose by treating with a cocktail of SEQ ID NOs 2,4,6,8 (PDX1, MafA, GLP-1R, and FDF21) and an epigenetic modulator. This claim language is problematic because it is known in the art that the administration of epigenetic modulators such as 5’-azacytidine in fact exacerbate and dysregulate hyperglycemic conditions leading to an increase in blood glucose (see discussion related to Ponard above and incorporated here). Furthermore, this claim language includes the broad category of “normalized” blood glucose, which would include normalizing hypoglycemic conditions (low blood glucose) as well as hyperglycemic conditions (high blood glucose). This claim language is problematic because the Applicant has only demonstrated that their method can lower blood glucose in mice, not elevate blood glucose levels. Finally, claim 11 broadly recites that the amino acid sequence is 95% identical to SEQ ID NOs 2,4,6,8. This claim language is problematic because it broadly includes mutations in the recited amino acids which were not tested.
With regards to the specific subject matter in the specification as it relates to claim 11, the Applicant offers Example 1. Example 1 demonstrates that a diabetic model of mice were administered a cocktail of SEQ ID NOs 2,4,6,8 in combination with 5’-azatytidine to reduce blood glucose levels (Example 1, page 14-15). The Applicant does not offer guidance with a mutational strategy to determine functional variants of SEQ ID NOs 2,4,6,8 which would render functional components of the recited reprogramming cocktail. The Applicant has only tested amino acids with 100% identity to SEQ ID NOs 2,4,6,8. Furthermore, the Applicant has only reduced to practice that their method is functional in mice, where the art teaches that such epigenetic modulators work to exacerbate hyperglycemic blood glucose levels in humans (see discussion of Ponard, above). Finally, the Applicant does not show that hypoglycemia, an abnormal blood glucose level, could be addressed using the recited method, which only shows that blood glucose can be lowered in the mouse model and not raised. In this respect, the practitioner is a priori not enabled to treat hypoglycemia, as the method has only been shown to lower blood glucose levels and not raise them.
State of the Art
As an initial matter, the Applicant’s data only show that high blood glucose can be lowered, but not that hypoglycemia can be treated using an epigenetic modulator cocktail also including amino acids represented by SEQ ID NOs 2,4,6,8 (Example 1). As such, the practitioner is a priori not enabled to treat hypoglycemia using the recited method because the application does not show that the method can increase blood glucose levels.
Regarding the genus of amino acids recited, where amino acids comprising up to 5% mutations are encompassed in the claims for SEQ ID NOs 2,4,6,8, it is known in the art that such mutations can have drastic and unpredictable effects on the structure and function of amino acids. For instance, Bourdonnaye (de La Bourdonnaye G et al. Comput Struct Biotechnol J. 2024 Feb 7;23:942-951) is a research article that focuses on engineering variants of FGF21 (i.e., SEQ ID NO: 8). Bourdonnaye teaches that presently, FGF21 has several characteristics which make it unfavorable for use in therapeutics, including its susceptibility to heat, proteolytic, and acid-mediated degradation as well as its susceptibility to kidney clearance (Abstract). Bourdonnaye teaches that such barriers must be overcome in order to effectively use FGF21 (Abstract). Bourdonnaye teaches that in order to overcome these therapeutic barriers, FGF21 must be engineered in order to increase its thermostability, where such engineering requires rational design and experimentation (Abstract). Bourdonnaye teaches that “rational engineering of stable FGF21 variants” can be made “by introducing a disulfide bridge in the protein core and truncating four amino acids on the N-terminus,” (page 2, left column, second paragraph). Bourdonnaye teaches that rational single-point mutations must be tested empirically to determine their efficacy and characteristics (Discussion, first paragraph). Thus, Bourdonnaye teaches that, in general, there are known problems associated with introducing FGF21 into therapeutic applications, where furthermore such issues with the protein must be addressed using strategies such as single-point mutation where such proteins generated must be tested empirically (Abstract, Discussion). Furthermore, given that it is know that such issues exist with the introduction of proteins, where furthermore point mutations are known to alter their stability and characteristics, the analysis of Bourdonnaye also applies to the remaining amino acids SEQ ID NOs 2,4, and 6.
Experimental Burden
The practitioner is unduly burdened with experiment in order to carry out the presently recited method. For instance the practitioner would at the very least be required to engineer, test, and design individual point mutations for each of the recited SEQ ID NOs 2,4,6,8, to determine functional embodiments of each protein. The Applicant has provided no guidance with respect to which domains or point mutations can be modified in order to retain protein functionality, or which domains may render unexpected results such as improved thermostability for the in vivo use of such proteins. Thus, the practitioner would burdened by having to identity such regions and test such mutants in order to identify such useful variants.
Conclusion
The practitioner is not enabled for the presently recited method claims because the scope of what they are claiming can not be practiced without undue experimental burden placed on the practitioner, where furthermore it is known in the art that the use of epigenetic modulators such as 5’-azacytidine is not likely to result in expected outcomes, as it can lead to exacerbated hyperglycemia requiring increased monitoring and insulin administration in human subjects. Furthermore, diabetes is a highly complex disease involving multiple genes and environmental factors, where specific methylation targets must be empirically tested and defined, where furthermore new untested targets are still being discovered (see above). Additionally, the application does not provide sufficient support to test variations in SEQ ID NOs 2,4,6,8 without placing undue experimental burden on the practitioner.
Claim Rejections - 35 USC § 102
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.
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-5 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Zhang (Zhang L et al. Kidney Int. 2017 Jul;92(1):140-153).
Regarding claim 1, Zhang teaches the treatment of diabetes using a DNA methylation inhibitor in mice (Abstract).
Regarding claim 2, given that Zhang has already taught the administration of a DNA methylation inhibitor to treat diabetes (i.e., an epigenetic modulator), the administration of said epigenetic modulator would co-occur with the treatment of diabetes and its related condition, such as hyperglycemia. Thus, the method of Zhang to treat diabetes with an epigenetic modulator broadly applies to the disease state and symptoms of a diabetic condition including hyperglycemia.
Regarding claim 3, as Zhang has taught that the administration of an epigenetic modulator to treat diabetic conditions (Abstract), a practitioner could immediately envision that such a treatment would be further accompanied by other therapeutic agents to treat diabetes (e.g., routine insulin administration to manage diabetes). Furthermore, Zhang also teaches that “drugs,” plural, can be used to treat diabetic nephrophathy (DN), and therefore teaches the epigenetic modulator to be used with other therapeutic agents (i.e., “drugs,” page 150, left column, third paragraph).
Regarding claims 4-5, Zhang teaches that the epigenetic modulator was the methyltransferase inhibitor 5’azacytidine (Abstract).
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.
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 6-8 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang L et al. Kidney Int. 2017 Jul;92(1):140-153) and further in view of Choudhury (Choudhury SR et al. Oncotarget. 2016 Jul 19;7(29):46545-46556) and Yankova (Karachanak-Yankova S et al. Balkan J Med Genet. 2016 Jul 9;18(2):15-24).
Regarding claims 6-7, the teachings related to Zhang are discussed in the 102 rejection above and incorporated here. Zhang teaches a method of treating diabetes with an epigenetic modulator in mice (Abstract).
Zhang does not teach that the epigenetic modulator is targeted to specific genes for modulation, where the modulator is the dCas9-TET1CD system for targeted demethylation.
Choudhury is a research article which teaches the dCas9-TET1CD fusion system for the targeted demethylation of promoter regions (genes, Title, Abstract, and throughout). Choudhury and Zhang therefore directly overlap because they are both related to demethylation strategies in order to treat diseases. Choudhury teaches that targeted demetheylation of specific genes to activate their expression is a useful therapeutic strategy (page 46546, right column, first paragraph). Choudhury teaches that their method of targeted demethylation can be expanded to target different genes (page 46546, right column, first paragraph). Choudhury teaches that genes such as BRCA1 were targeted (Title, throughout).
Furthermore, Yankova is a research article that focuses on epigenetic modifications in patients with Type 2 diabetes (Title, Abstract, and throughout). Yankova further teaches that tumor suppressor genes such as BRCA1 and TP53 are hypermethylated in type 2 diabetics, along with other genes such as PrdX2 and SCARA3 (Abstract). Thus, Yankova teaches that the BRCA1 gene, like Choudhury, is also hypermethylated in diabetes (Abstract). Yankova therefore teaches that it is known that type 2 diabetes is associated with specific target genes that are hypermethylated such as TP53 (Abstract, throughout).
It would have been obvious to a person of ordinary skill in the art before the effective filing date to modify the epigenetic modulator 5-azacytidine taught by Zhang to include an epigenetic modulator such as the dCas9-TET1CD system taught by Choudhury to target TP53 as taught by Yankova, as such a combination is the simple substitution of one known element for another with predictable results. In the present case, the concept of targeting epigenetic markers by using epigenetic modulators that target specific genes such as dCas9-TET1CD was already known and reduced to practice, per Choudhury. The practitioner would therefore simply substitute the known epigenetic modulator 5’-azacytidine (Zhang) for gene specific epigenetic modulators such as dCas9-TET1CD (Choudhury). Furthermore, the practitioner would be motivated to adopt the strategy of Choudhury to target diabetes-induced epigenetic modifications because 1) Choudhury teaches that their method is useful as a therapeutic which can be modified and adopted to target other genes and 2) Yankova teaches known epigenetic gene targets which are associated with diabetes, such as TP53. Thus, the art teaches targeting of TP53 for demethylation in a diabetic context.
Regarding claim 8, Choudhury teaches that the demethylation of targeted genomic regions is the promoter (Title). Choudhury teaches polynucleotides encoding the dCas9-TET1CD fusion and gene targeting guide RNA, and that such designs are modular (e.g., Figure 1). Choudhury teaches that transfection of cells can be optimized and modified (Figure 1); the practitioner of ordinary skill could therefore immediately envision and/or arrive at through routine laboratory optimization a co-transfection of the guide RNA and dCas9-TET1CD system (page 46552, right column, first paragraph). Furthermore, given that Zhang and Choudhury teach the therapeutic application of their constructs, the practitioner would immediately understand that such constructs would be administered to the tissue of a diabetic (Zhang/Choudhury Abstracts).
Claim 9 and 14-16 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang L et al. Kidney Int. 2017 Jul;92(1):140-153) in view of Choudhury (Choudhury SR et al. Oncotarget. 2016 Jul 19;7(29):46545-46556) and Yankova (Karachanak-Yankova S et al. Balkan J Med Genet. 2016 Jul 9;18(2):15-24), as applied to claims 6-8, above, and further in view of Dekker (Dekker et al. Transl Res. 2019 Feb;204:39-50).
The teachings of Zhang, Choudhury, and Yankova are discussed above and incorporated here. In summary, the teachings of Zhang in view of Choudhury and Yankova render obvious a method of targeting diabetes-induced epigenetic modifications using modulators including the dCas9-TET1CD system to target specific epigenetically modified target genes associated with diabetes such as TP53 (see rejection of claim 6, above).
Zhang, Choudhury, and Yankova do not specifically teach that the gRNA and dCas9-TET1CD are transfected at into tissues adjacent to a chronic wound.
Dekker is a research article focused on targeting epigenetic mechanisms in diabetic wound healing (Title, Abstract, and throughout). Dekker teaches that:
“Several recent studies suggest that altered epigenetic regulation of both immune and structural cells in wounds may influence cell phenotypes and healing, particularly in pathologic states, such as diabetes,” (Abstract).
Dekker therefore teaches that it is known in the art that altered epigenetic states in wound cells are associated with pathogenic states such as diabetes and therefore teaches that such tissue/wound sites are known targets for treatment (Abstract).
It would have been obvious to a person of ordinary skill in the art to modify the teachings of Zhang, Choudhury, and Yankova, who render obvious the application of epigenetic regulators to target specific sites in a diabetics genome using the dCas9-TET1CD system targeting TP53, to apply the strategy to wound tissue because such a combination is the simple combination of known prior art elements with predictable success. In the present case, the practitioner would apply the technique and therapeutic strategy rendered obvious by Zhang, Choudhury, and Yankova to a wound as taught by Dekker. Furthermore, the practitioner would be motivated to apply the dCas9-TET1CD targeting strategy to wounds because Dekker teaches that it is known that epigenetic dysregulation is associated with pathological wound states of diabetics. The practitioner would therefore be motivated to target the wounds of diabetics because it is known that such wound areas comprise epigenetic dysregulation, which is the basis of the treatment strategy rendered obvious by Zhang/Choudury/Yankova, where furthermore specific targets such as TP53 are known, per Yankova.
Regarding claim 14, claim 14 is broadly drawn to method of wound repair in diabetes by administration of a cocktail to target the demethylation of genes involved in wound repair, where “cocktail” can most broadly be interpreted to mean simply a combination of a guide RNA and Cas9-TET1CD complex. Such a cocktail is rendered obvious by the combination of Zhang, Choudhury, Yankova, and Dekker (see rejection of claim 9, above). Furthermore Dekker also teaches that:
“JMJD3 is necessary for normal keratinocyte differentiation by promoting expression of differentiation-associated genes, while the H4K20 methyltransferase, SETD8, promotes normal keratinocyte proliferation and differentiation. Taken together, these reports suggest that epigenetic regulators could prove to be viable therapeutic targets for treating wound healing in diabetes and other conditions, “(page 6, final paragraph).
Thus, Dekker teaches that multiple genes can be targeted for wound healing in a diabetic, where the genes are associated with epigenetic regulation (page 6, final paragraph). Given that Choudhury teaches that multiple gRNAs can target promoters and target different genes, where their system is taught to be modular, the practitioner would be further motivated to target multiple genes and/or a make a cocktail of targets to treat wound healing in diabetics to target known diabetes associated genes such as those taught by Yankova i.e., TP53, BRCA1, etc., (Choudhury page 46546, right column first paragraph, Figure 1, Dekker page 6 final paragraph, Yankova Abstract).
Regarding claim 15, Dekker teaches that the cells are keratinocytes (Abstract, page 6 final paragraph).
Regarding claim 16, Yankova teaches that TP53 is a gene which is dysregulated in diabetics and therefore teaches a reason to target TP53 with the epigenetic regulator rendered obvious (Yankova, Abstract).
Claims 10 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang L et al. Kidney Int. 2017 Jul;92(1):140-153) in view of Choudhury (Choudhury SR et al. Oncotarget. 2016 Jul 19;7(29):46545-46556), Yankova (Karachanak-Yankova S et al. Balkan J Med Genet. 2016 Jul 9;18(2):15-24), and Dekker (Dekker et al. Transl Res. 2019 Feb;204:39-50) as applied to claims 9 and 14-16 above, and further in view of Gallego-Perez (Gallego-Perez D et al. Nat Nanotechnol. 2017 Oct;12(10):974-970.
Regarding claims 10 and 17, The teachings of Zhang, Choudhury, Yankova, and Dekker are discussed above and incorporated here. Choudhury teaches that their constructs can be transfected into cells (Figure 1).
Zhang, Choudhury, Yankova, and Dekker do not teach that the tissue is transfected via nanotransfection.
Gallego-Perez is a research article that focuses on topical tissue nano-transfection (Title, Abstract, and throughout). Gallego-Perez teaches transfection (Abstract) and teaches transfecting a tissue via nanotransfection (page 974, left column, last paragraph). Gallego-Perez teaches that the tissue nano-transfection (TNT) approach allows direct cytosolic delivery of reprogramming factors by applying a highly intense and focused electric field through arrayed nanochannels (page 974, left column, last paragraph). Gallego-Perez teaches that their method is simple, and offers advantages such as direct topical administration for cell reprogramming that is safer than other methods (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date to modify the delivery methods of Zhang, Choudhury, Yankova, and Dekker, to include nanotransfection as taught by Gallego-Perez because Gallego-Perez teaches that such nanotransfection strategies have known advantages such as simplicity and safety, where such administration allows for direct transfection to a desired area.
Claims 11-13 are rejected under 35 U.S.C. 103 as being unpatentable over Gallego-Perez 2 (WO 2020028697 A1, published 2/6/2020) in view of Pan (Pan Y et al. J Cell Mol Med. 2019 Feb;23(2):1059-1071), Sasaki (Sasaki S et al. Diabetologia. 2015 Nov;58(11):2582-91) and Zhang (Zhang L et al. Kidney Int. 2017 Jul;92(1):140-153).
Regarding claim 11, Gallego-Perez 2 is a patent document which focuses on skin cells and tissue into insulin-producing tissue (Title, Abstract, and throughout). Gallego-Perez 2 therefore teaches that a method of reprogramming skin cells/tissue into insulin producing tissue/cells is known in the art (Title, throughout). Gallego-Perez 2 teaches a method of reprogramming skin cells to produce insulin in an in vivo mouse model, where the method comprises introducing a reprogramming composition comprising nucleic acid sequences encoding proteins PDX1 and Mafa (i.e., instant SEQ ID NOs 2 and 4, per the instant specification, Example 1, page 14, paragraph 7, and see Example 1 on page 23 and claim 6 of Gallego-Perez 2). Gallego-Perez 2 teaches that the method is used to treat diabetes, which reasonably includes stabilizing blood glucose levels (claim 12 of Galego-Perez 2). Gallego-Perez 2 teaches that the cells are transfected with a nanoelectroporation device, and therefore teaches that the method includes conditions that enhance cellular uptake (claim 11 of Gallego-Perez 2). Gallego-Perez 2 teaches that the reprogramming composition can comprise further factors such as Ng3 and Tcf3 (claim 1 of Gallego-Perez 2).
Gallego-Perez 2 does not teach that the method comprises contacting the skin tissue with an epigenetic modulator complex and that the reprogramming composition further comprises SEQ ID NOs 6 and 8 (i.e., GLP-1R and FDF21, per the instant specification at page 14, paragraph 7).
Pan is a research article which focuses on cellular factors associated with insulin production in cells and their relationship to diabetes (Title, Abstract, and throughout). Pan and Gallego-Perez 2 therefore overlap in subject matter because both teach diabetes and factors associated with insulin production in cells. Pan teaches that FGF21 (i.e., instant SEQ ID NO: 8) is known to be associated with insulin production in cells, and further that its overexpression enhances insulin production (Abstract). Pan teaches that their study was conducted in a mouse model (Abstract). Thus, FDF21 (instant SEQ ID NO: 8) and its overexpression in cells is known to be associated with an enhanced insulin production (Pan, Abstract). Furthermore, Pan teaches that overexpression of FGF21 enhanced expression of PDX1 and MafA genes, the factors taught by Gallego-Perez 2 (see section 3.4 of Pan).
Furthermore, Sasaki is a research article focused on reprogramming of in vivo cells for insulin production in mice (Title, Abstract, and throughout). Sasaki teaches that overexpression of GLP-1R (instant SEQ ID NO: 6) leads to an increase in insulin production in cells, and furthermore promotes PDX1 and MafA expression, transcription factors taught by Gallego-Perez 2 known to be expressed in insulin produced cells (Abstract).
Additionally, Zhang is a research article focused on methods of treating diabetes (Title, Abstract, and throughout). Zhang teaches that it is a known strategy to include epigenetic modulators to treat diabetes in mouse models, where the epigenetic modulator is 5’-azacytidine (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date to modify the skin tissue reprogramming strategy taught by Gallego-Perez 2 to which includes in the composition instant SEQ ID NOs 2 and 4 (PDX1 and MafA) to further include FGF21 (SEQ ID NO 8) as taught by Pan and GLP-1R (SEQ ID NO: 6) as taught by Sasaki, as such a combination is the simple combination of known prior art elements with predictable results. Furthermore, the combination is not simply combining elements; a practitioner would be motivated to combine the FGF21 and GLP-1R components because both of these are known to promote insulin production in cells in vivo, which was the stated goal of Gallego-Perez 2, where furthermore both FGF21 and GLP-1R were known to promote the expression of the same compositional elements taught by Gallego-Perez 2 (i.e., PDX1 and MafA). There is therefore a strong motivation to include the known elements as taught in the art. Furthermore, the strategy of including an epigenetic modulator is obvious as taught by Zhang, who teaches that such compounds are useful in the treatment of diabetic conditions in mice, where Zhang teaches 5’-azatydine. Furthermore, each of Gallego-Perez 2, Sasaki, Zhang, and Pan use mouse models, where the outcome is predictable because the same model organisms are being used in each study.
Regarding claim 12, Zhang teaches that the epigenetic modulator is 5’azacytidine (Abstract).
Regarding claim 13, Gallego-Perez 2 teaches that the compounds can be introduced by nanotransfection (claim 10). Furthermore, a practitioner could at once envision administering the epigenetic modulator at the same site to co-administer the recited cocktail.
Conclusion
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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