DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
2. Applicant's election with traverse of Group I and the species of the combination of LMR 1, 2, 3, 4 and 5 in the reply filed on 17 February 2026 is acknowledged. The traversal is on the ground(s) that all of the claims use low methylation regions (LMRs) as an indicator for gut inflammation in avian subjects and all of the LMRs are equally effective. This is not found persuasive because, as set forth in the restriction requirement of 17 December 2025 the claims of Group I and II do not in fact share the same corresponding technical feature. Claim 1 of Group I and claim 12 of Group II are drawn to different processes, involving different method steps and having different objectives and outcomes. Secondly, regions of low methylation were known in the prior art and thereby this is not a special technical feature. For example, Luo et al (Frontiers in Genetics. 2012. 3(2): p. 1-15) teaches regions in the avian genome which have low levels of methylation and in which methylation levels are correlated with resistance or susceptibility to Markek’s disease virus (MDV) infection (e.g., abstract; p. 2, col. 2, and Tables A3 and A4). Regarding the recited species of LMRs, the LMRs do not share a special technical feature because each of the LMRs have a different chemical structure. That is, each of the LMRs occur at different locations in the avian genome and have different nucleotide sequences, including different sequences flanking the CpGs. The LMRs do not have both a "common property or activity" and a common structure essential to that property or activity as would be required to show that the inventions are "of a similar nature."
The requirement is still deemed proper and is therefore made FINAL.
Claim Status
3. Claims 1-15 are pending.
Claims 12-15 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim 7 is withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 1-6 and 8-11 read on the elected invention and have been examined herein. It is noted that claim 6 encompasses the non-elected subject matter of LMRs other than the elected combination of LMRs 1-5. Prior to the allowance of claims, any non-elected subject matter which has not been rejoined with the elected subject matter will be required to be removed from the claims.
Specification
4. The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code – see, for example, page 12, lines 7, 8 and 21-22. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code, such as “www.”. See MPEP § 608.01.
5. The use of the terms Illumina™ (at para [0051]; note that paragraph numbering herein is with respect to the published application) and “Random Forest® Analysis” (e.g., para [0045]) which are trade names or marks used in commerce, have been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
These are examples of trademarks that are recited in the specification. The specification should be reviewed for any additional trademarks or trade names and the terms should be accompanied by their generic terminology and capitalized or where appropriate accompanied by a proper symbol.
Claim Objections
6. Claims 1-6 and 8-11 are objected to because of the following informalities: Claims 1-6 and 8-11 are objected to because the claims should recite an “and” prior to “b.” in claim 1 so that the claims will be ‘close-ended’ (i.e., since “b.” is the final step of the claims). Appropriate correction is required.
Claim Rejections - 35 USC § 101
7. 35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-6 and 8-11 are rejected under 35 U.S.C. 101 because the claimed invention is directed to the judicial exception of a law of nature / natural phenomenon, and/or an abstract idea without significantly more. The judicial exception is not integrated into a practical application and the claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception for the reasons that follow.
Applicant' s attention is directed to MPEP 2106 “Patent Subject Matter Eligibility” which discusses the Alice/Mayo two-part test for evaluating subject matter eligibility.
Regarding Step 1 of the subject matter eligibility test set forth at MPEP 2106III, the claims are directed to the statutory category of a process.
Regarding Step 2A, prong one, the claims recite the judicial exception of a law of nature. The claims recite the correlation between the level of methylation of low-methylated regions (LMRs) and gut inflammation of an avian subject or group of avian subjects. Note that the claims are drawn to a method for classifying gut inflammation in an avian subject or group of avian subjects and recite “wherein when the test average methylation level is substantially similar to the reference average methylation level, then the test subject has a negative gut inflammation status and when the test average methylation level is different from the reference average methylation level then the test subject has a positive gut inflammation status” (see claim 1). As in Mayo Collaborative Services v. Prometheus, the recited relationship is a natural phenomenon that exists apart from any human action. See also Cleveland Clinic Foundation v. True Health Diagnostic, LLC, 2018-1218 (Fed Cir. 2019) which states that “The re-phrasing of the claims does not make them less directed to a natural law.”
The claims also recite the judicial exception of an abstract idea and particularly mental processes.
MPEP 2106.04(a) states that the enumerated groupings of abstract ideas include:
“1) Mathematical concepts – mathematical relationships, mathematical formulas or equations, mathematical calculations (see MPEP § 2106.04(a)(2), subsection I);…
3) Mental processes – concepts performed in the human mind (including an observation, evaluation, judgment, opinion) (see MPEP § 2106.04(a)(2), subsection III).”
The claims require “classifying” gut inflammation (claim 1) and “gut inflammation status is further classified” (claim 3). Neither the specification nor the claims set forth a limiting definition for "classifying" and the claims do not set forth how “classifying” is accomplished. The broadest reasonable interpretation of the classifying steps is that the steps may be accomplished by critical thinking processes in which the classification is made based on a comparison of the methylation level of a test sample and a reference level. Such classifying is thereby an abstract idea / process.
The claims also require performing a step of "comparing" methylation levels. Neither the specification nor the claims set forth a limiting definition for "comparing" and the claims do not set forth how comparing is accomplished. The broadest reasonable interpretation of the “comparing” step is that this step may be accomplished by critical thinking processes. Such “comparing” thereby encompasses only an abstract idea / process. The classifying may also be accomplished verbally. Such verbal communication is also abstract, having no particular concrete or tangible form.
Further, the “diagnosing” step is merely a restatement of the natural law / natural phenomenon – i.e., the correlation between the risk of glioblastoma or aggressive glioblastoma and the microsatellite profile. The “diagnosing” step thereby does not add something significantly more to the natural law / natural phenomenon.
Regarding Step 2A, prong two, having determined that the claims recite a judicial exception, it is then determined whether the claims recite additional elements that integrate the judicial exception into a practical application.
Herein, the claims do not recite additional steps or elements that integrate the recited judicial exceptions into a practical application of the exception(s). The additionally recited step of determining the methylation level of LMRs in a gut test sample is part of the data gathering process necessary to observe the judicial exception. This step does not practically apply the judicial exception.
Regarding Step 2B, the next question is whether the remaining elements/steps – i.e., the non-patent-ineligible elements/steps - either in isolation or combination, amount to significantly more than the judicial exception.
Herein, the claims as a whole are not considered to recite any additional steps or elements that amount to significantly more than routine and conventional activity and do not add something “significantly more” so as to render the claims patent-eligible. The additionally recited step of determining methylation level of LMRs was well-known, routine and conventional in the prior art.
This finding is evidenced by the teachings in the specification. For instance, para [0020] (paragraph numbering herein is with respect to the published application) of the specification states: “Measurement of the levels of differential methylation may be done by a variety of ways known to those skilled in the art.” Further, para [0036] states:
“any method known in the art may be used to identify or detect LMRs in the genomic DNA. Well known methods include using programmes such as MethylSeekR.”
See also MPEP 2106.05(d) II which states that:
The courts have recognized the following laboratory techniques as well-understood, routine, conventional activity in the life science arts when they are claimed in a merely generic manner (e.g., at a high level of generality) or as insignificant extra-solution activity.
i. Determining the level of a biomarker in blood by any means, Mayo, 566 U.S. at 79, 101 USPQ2d at 1968; Cleveland Clinic Foundation v. True Health Diagnostics, LLC, 859 F.3d 1352, 1362, 123 USPQ2d 1081, 1088 (Fed. Cir. 2017);
ii. Using polymerase chain reaction to amplify and detect DNA, Genetic Techs. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016); Ariosa Diagnostics, Inc. v. Sequenom, Inc., 788 F.3d 1371, 1377, 115 USPQ2d 1152, 1157 (Fed. Cir. 2015);
iii. Detecting DNA or enzymes in a sample, Sequenom, 788 F.3d at 1377-78, 115 USPQ2d at 1157); Cleveland Clinic Foundation 859 F.3d at 1362, 123 USPQ2d at 1088 (Fed. Cir. 2017); …
v. Analyzing DNA to provide sequence information or detect allelic variants, Genetic Techs., 818 F.3d at 1377; 118 USPQ2d at 1546; …
vii. Amplifying and sequencing nucleic acid sequences, University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 764, 113 USPQ2d 1241, 1247 (Fed. Cir. 2014); and
viii. Hybridizing a gene probe, Ambry Genetics, 774 F.3d at 764, 113 USPQ2d at 1247.
Note that while claim 6 recite detecting the methylation level of particular LMRs, the identity of the LMR is part of the judicial exception and not something in addition to the recited judicial exceptions. The claims do not require using a particular non-conventional reagent, such as a particular, non-conventional probe or primer consisting of or comprising a specific nucleotide sequence to detect the methylation level so as to add something ‘significantly more’ to the recited judicial exceptions.
In Mayo v. Prometheus, the Supreme Court stated: "[t]o put the matter more succinctly, the claims inform a relevant audience about certain laws of nature; any additional steps consist of well understood, routine, conventional activity already engaged in by the scientific community; and those steps, when viewed as a whole, add nothing significant beyond the sum of their parts taken separately."
This is similar to the present situation wherein the additional steps and elements are recited at a high degree of generality and are all routine, well understood and conventional in the prior art. The recited steps and elements do not provide the inventive concept necessary to render the claims patent eligible. See also Genetic Technologies Ltd. v. Merial L.L.C. 818 F.3d at 1377, 1379 (Fed. Cir. 2016).
For the reasons set forth above, when the claims are considered as a whole, the claims are not considered to recite something significantly more than a judicial exception and thereby are not directed to patent eligible subject matter.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
8. 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 1-6 and 8-11 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1-6 and 8-11 are indefinite over the recitation of “classifying gut inflammation status of an avian subject or of a group of avian subjects to be tested, an avian test subject, the method comprising” because it is not clear as to what is meant by this phrase. Note that the claims go on to recite obtaining the sample “from the avian test subject,” and thereby specifically reference the avian test subject but do not specifically later refer to the avian subject or group of avian subjects to be tested.
Claims 1-6 and 8-11 are indefinite over the recitation of “substantially similar.” This phrase is not clearly defined in the specification or the claims. In the context of the claims, it is unclear as to the degree of difference between the test average methylation level and the reference average methylation level that would indicate that the average methylation levels are “substantially similar” as compared to the methylation levels being “different.” It is noted that the specification at para [0012] defines “significantly similar”; however, “significantly similar” is not the same terminology as “substantially similar. Also the definition for “significantly similar” - i.e., “in the context of the disclosure is a similarity observed by either statistical means (i.e. bioinformatics) or by empiric observation” does not provide a fixed meaning for what is encompassed by “significantly similar” since an empiric observation is subjective and no particular statistical means is required. Accordingly, to any extent that Applicant would rely on the disclosure in the specification regarding “significantly similar” to define “substantially similar,” the artisan would not know when two methylation levels are “significantly similar” and when they are not “significantly similar.”
Claim 5 contains the trademark/trade name “Random Forest® Analysis.” MPEP 2173.05 states “If the trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of the 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph. Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982).”The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe and, accordingly, the identification/description is indefinite.
Claim 6 is indefinite over the recitation of the chromosomal positions at which the LMR is located (i.e., the “Start” and “End” positions). Note that the claims do not recite a particular reference genome for the chromosomal positions. The nucleotide numbering of chromosomes varies with the reference chromosome that is utilized and thereby there is no fixed meaning for what constitutes, e.g., position "4935642” in chromosome 1. The nucleotide numbering of chromosomes also varies significantly between species, including the diverse avian species encompassed by the claims, such as chickens, turkeys, ducks, woodpeckers, herons etc. The specification indicates that the chromosome positions were determined relative to the Gallus gallus genome assembly version 5.0 (available via url: <ebi.ac.uk/ena/data/view/GCA_000002315.3>) reference sequence. However, the specification does not indicate that all chromosome positions disclosed therein are with respect to the Gallus gallus genome assembly version 5.0. Nor do the claims do not recite that the avian is limited to Gallus gallus and that the chromosome positions are defined relative to the Gallus gallus reference genome assembly version 5.0. As such, the recitation of the chromosomal positions are without context and it is unclear as to what constitutes the particular LMRs with respect to Gallus gallus or any other avian species.
Regarding claim 10, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim Rejections - 35 USC § 112(a)– Written Description
9. The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-6 and 8-11 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a written description rejection.
In analyzing the claims for compliance with the written description requirements of 35 U.S.C. 112, first paragraph, a determination is made as to whether the specification contains a written description sufficient to show they had possession of the full scope of their claimed invention at the time the application was filed.
For claims drawn to a genus, MPEP § 2163 states:
“The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice (see i)(A) above), reduction to drawings (see i)(B) above), or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the inventor was in possession of the claimed genus (see i)(C) above). See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. See Juno Therapeutics, Inc. v. Kite Pharma, Inc., 10 F.4th 1330, 1337, 2021 USPQ2d 893 (Fed. Cir. 2021) ( "[T]he written description must lead a person of ordinary skill in the art to understand that the inventor possessed the entire scope of the claimed invention. Ariad, 598 F.3d at 1353–54 ('[T]he purpose of the written description requirement is to ensure that the scope of the right to exclude, as set forth in the claims, does not overreach the scope of the inventor's contribution to the field of art as described in the patent specification.' (internal quotation marks omitted).").”
MPEP § 2163 goes on to state:
“An adequate written description of a chemical invention also requires a precise definition, such as by structure, formula, chemical name, or physical properties, and not merely a wish or plan for obtaining the chemical invention claimed. See, e.g., Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 927, 69 USPQ2d 1886, 1894-95 (Fed. Cir. 2004).”
A. With respect to the elected species, claim 6 is drawn to a method for classifying the gut inflammation status of an avian subject or of a group of avian subjects to be tested, wherein the method comprises determining the methylation level of LMR 1-5 listed in claim 6:
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However, the specification does not adequately describe these LMRs. It is not clear as to what constitutes the start and end positions of the LMRs because the claims do not set forth a reference genome so that the context of the nucleotide positions is clear. The nucleotide numbering of chromosomes varies with the reference chromosome that is utilized and thereby there is no fixed meaning for what constitutes each of the start and end positions for the LMRs. The specification indicates that the chromosome positions were determined for broiler chickens relative to the Gallus gallus (Red jungle fowl) genome assembly version 5.0 (available via url: <ebi.ac.uk/ena/data/view/GCA_000002315.3>) reference sequence. However, the claims do not recite that the avian is limited to broiler chickens / a Gallus gallus species and that the chromosome positions are defined relative to the Gallus gallus reference genome assembly version 5.0.
Further, the nucleotide numbering of chromosomes varies significantly between species, including the diverse avian species encompassed by the claims, such as chickens, turkeys, ducks, woodpeckers, herons etc. The specification does not teach that the chromosome positions for the LMR 1-5 recited in claim 6 are present in a representative number of diverse avian species.
Accordingly, the specification does not provide an adequate description of the elected LMRs 1-5 in Gallus gallus or a representative number of alternative avian species.
B. Secondly, claims 1-5 and 8-11 are dawn to a method for classifying the gut inflammation status of an avian subject or of a group of avian subjects to be tested, wherein the method comprises determining the methylation level of LMRs which are not defined in terms of their overall structure or any other relevant structural characteristics. Claim 3 further requires classifying the gut inflammation as severely inflamed, moderately inflamed, or weakly inflamed based on the average methylation level based on the methylation level of the LMRs. However, again the LMRs are not defined in terms of their complete structure or any other relevant identifying characteristics. Claim 2 defines the LMRs as having an average methylation ranging from 10% to 50%;being a region of low CG density; a region enriched for Histone H3 monomethylated at lysine 4 (H3K4mel1), DNase I hypersensitive sites (DHSs) and transcriptional coactivators CREB binding protein (CPB) and p300; primarily located distal to promoters in intergenic or intronic regions; and having no single nucleotide polymorphisms (SNPs) in any of the CpG positions. However, these functional properties do not provide a clear structure for the LMRs.
The genus of LMRs encompassed by the claims is further enlarged by the fact that the claims encompass methods that detect the methylation level of the LMRs in any avian. As set forth in the specification (para [0051], the avian subject may be “chickens, turkeys, ducks and geese.” Avian species also include diverse organisms owls, parrots, hummingbirds, woodpeckers, herons, bluebirds etc.
Accordingly, the claims encompass detecting the methylation level of a significantly large genus of LMRs in any avian species as indicative of gut inflammation per se, or severely inflamed, moderately inflamed, or weakly inflamed gut inflammation (claim 3).
However, the specification (Table 3) teaches only 15 LMRs that were used to classify the presence of gut inflammation in broiler chickens. As discussed above, these LMRs have only been described in terms of their chromosome positions with respect to Gallus gallus reference genome version 5.0.
The specification teaches that 13 of these LMRs are required for accurate detection of gut inflammation. That is the specification (para [0067] states:
“FIG. 1 : Classification error of an iterative random forest 3-fold cross-validation with 100 LMRs as starting input, applying the command rfcv of the R package RandomForest. The figure shows that the error becomes very small starting at 13 LMRs. At 6 LMRs the predictive power decreases and the error increases significantly.”
At p. 8 of the specification and Table 4, LMR16-LMR30 are disclosed which appear to have been selected based on the maximum methylation differences between the LMRs control and inflamed samples (e.g., para [0082]).
However, the disclosure of the LMRs, which are to be used in combinations, rather than as individual LMRs to detect gut inflammation (see, e.g., Fig. 1 and para [0067]), does not appear to constitute a representative number of LMRs in the broadly claimed genus. As shown in Fig. 2, the size of the genus of LMRs in general is at least 67,651 for broiler chickens.
Accordingly, the specification has not described by its complete structure or by other relevant identifying characteristics a representative number of species within the broadly claimed genus.
It is noted that the specification also teaches the general methodology for performing assays to identify LMRs and to detect the level of methylation of LMRs. However, possession may not be shown by merely describing how to obtain possession of members of the claimed genus or how to identify their common structural features.
Thereby, a showing of how to potentially identify other LMRs whose methylation status is correlated with gut inflammation is not sufficient to establish that Applicants were in possession of the invention as broadly claimed.
Additionally, the specification does not disclose a clear structure-function relationship between the claimed genus of LMRs and the property of having a methylation status at the LMR that is indicative of gut inflammation in a representative number of avians. No common structure has been disclosed to identify those members of the claimed genus of LMRs whose differential methylation is correlated with gut inflammation.
With respect to the present invention, there is no record or description which would demonstrate conception of a representative number of LMRs whose methylation level is indicative of gut inflammation or the degree of inflammation (claim 3). Therefore, the claims fail to meet the written description requirement because the claims encompass a significantly large genus of low-methylated regions diagnostic of gut inflammation which are not described in the specification.
Claim Rejections - 35 USC § 112(a) - Enablement
10. Claims 1-6 and 8-11 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 methods for classifying gut inflammation of a chicken or group of chickens to be tested wherein the methods comprise performing the method of claim 1 wherein the methylation level of each of LMR1-LMR15 recited in claim 6 are detected and compared to the methylation level of the LMRs of at least one gut tissue sample from a chicken not having gut inflammation and wherein the chromosome start and end positions of LMR 1-LMR 15 are relative to the Gallus gallus genome assembly version 5.0,
does not reasonably provide enablement for methods in which the subject is any avian; methods in which the methylation level of any LMR is detected as indicative of gut inflammation; or methods wherein only LMR 1-5 of claim 6 are detected as indicative of the classification of gut inflammation and the chromosome positions of the LMRs are not defined in terms of a reference genome. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
The following factors have been considered in formulating this rejection (In re Wands, 858F.2d 731, 8 USPQ2d 1400 (Fed. Cir. 1988): the breadth of the claims, the nature of the invention, the state of the prior art, the relative skill of those in the art, the predictability or unpredictability of the art, the amount of direction or guidance presented, the presence or absence of working examples of the invention and the quantity of experimentation necessary.
The present claims encompass methods for classifying gut inflammation in an avian test subject or a group of avian test subjects. The methods comprise: (a) determining a test average methylation level of a panel of pre-selected Low-Methylated Regions (LMRs) in isolated genomic DNA from a gut test sample derived from the avian test subject; and (b) comparing the test average methylation level obtained in step (a) with a reference average methylation level of the same panel of LMRs in genomic DNA isolated from at least one avian gut sample having a negative gut inflammation status, wherein when the test average methylation level is substantially similar to the reference average methylation level, then the test subject has a negative gut inflammation status and when the test average methylation level is different from the reference average methylation level then the test subject has a positive gut inflammation status.
Claims 1-5 and 8-11 do not define the panel of LMRs in terms of any specific structural properties. Claim 2 defines the LMRs as having an average methylation ranging from 10% to 50%;being a region of low CG density; a region enriched for Histone H3 monomethylated at lysine 4 (H3K4mel1), DNase I hypersensitive sites (DHSs) and transcriptional coactivators CREB binding protein (CPB) and p300; primarily located distal to promoters in intergenic or intronic regions; and having no single nucleotide polymorphisms (SNPs) in any of the CpG positions. However, these functional properties do not provide a clear structure for the LMRs.
With respect to claim 6 and the elected species of LMR1-5, it is unclear as to what is encompassed by the particular LMRs because the claim does not set forth a reference genome so that the context of the chromosome positions is clear. The nucleotide numbering of chromosomes varies with the reference chromosome that is utilized and thereby there is no fixed meaning for what constitutes each of the start and end positions for the LMRs. The specification indicates that the chromosome positions were determined for broiler chickens relative to the Gallus gallus (Red jungle fowl) genome assembly version 5.0 (available via url: <ebi.ac.uk/ena/data/view/GCA_000002315.3>) reference sequence. However, it is not clearly stated that all chromosome positions recited in the specification are with respect to this reference genome and claim 6 does not recite that the chromosome positions listed therein are with respect to this reference genome assembly.
Further, the claims encompass methods in which the test subject or group of test subjects is any avian species. The claims thereby encompass methods that determine the methylation status of the LMRs in such diverse species as turkeys, ducks, woodpeckers, herons, owls, chickadees etc., as well as chickens. However, nucleotide numbering of chromosomes varies significantly between species. The specification does not teach that the chromosome positions for the LMR 1-5 recited in claim 6 are the same in a representative number of diverse avian species.
Moreover, all results provided in the specification are with respect to broiler chickens. There is no showing that LMR1-5 or any of the other LMRs disclosed in the specification are present in a representative number of the diverse avian species encompassed by the claims and that these LMRs are differentially methylated and correlated with gut inflammation or the degree of gut inflammation in a representative number of diverse avian species.
Accordingly, the claims encompass detecting the methylation level of a significantly large genus of LMRs in any avian species as indicative of gut inflammation per se, or severely inflamed, moderately inflamed, or weakly inflamed gut inflammation (claim 3).
However, the specification (Table 3) teaches only the 15 LMRs of claim 6 which are used as a combination to classify the presence of gut inflammation in broiler chickens. As discussed above, these LMRs have only been described in terms of their chromosome positions with respect to Gallus gallus reference genome version 5.0.
At p. 8 of the specification and Table 4, LMR16-LMR30 are also disclosed which appear to have been selected based on the maximum methylation differences between the LMRs control and inflamed samples (e.g., para [0082]).
The specification teaches that 13 of LMR1-15 are required for accurate detection of gut inflammation. That is the specification (para [0067] states:
“FIG. 1 : Classification error of an iterative random forest 3-fold cross-validation with 100 LMRs as starting input, applying the command rfcv of the R package RandomForest. The figure shows that the error becomes very small starting at 13 LMRs. At 6 LMRs the predictive power decreases and the error increases significantly.”
Thus, the teachings in the specification indicate that only 5 LMRs, including LMR1-LMR5 of claim 6, is not sufficient to accurately classify cut inflammation.
There is a high level of unpredictability in the art of diagnosing a phenotype, such as gut inflammation of an avian subject, based on methylation status.
As discussed above, the teachings in the specification evidnece this level of unpredictability in that the specification teaches that a combination of 6 of LMR1-15 is not sufficient to determine gut inflammation without error (Fig. 2 and para [0068]). The teachings in the specification also establish that the identity of LMRs that are correlated with gut inflammation can only be identified by trial and error experimentation. Note that the specification and Figure 1 show that while 67,651 LMRs were found to be present in the genome of broiler chickens, the combination of 13 of these LMRs “resulted in non-zero classification error” and a combination of 15 of the LMRS is “sufficient for a close-to-zero classification.”
Additionally, the unpredictability of extrapolating the results from one avian species to others is supported by the fact that the sequence of chromosomes varies significantly between species
However, all results presented in the specification were obtained in human subjects. There is no showing in the specification that the IL28B promoter and other non-coding regions are differentially methylated in a representative number of non-human subjects or that the elected sequence of SEQ ID NO: 2 is present in a representative number of non-human subjects and is differentially methylated in response to a direct-acting antiviral therapy.
The art of extrapolating methylation status results from one animal to other animals is highly unpredictable. For example, Ehrlich et al. (Oncogene 2002. 21: 5400-5413) teaches that there are considerable differences in the amounts and distribution of DNA methylation among different vertebrate tissues because DNA methylation is not only species-specific but also tissue-specific (p. 5400 last paragraph). Similarly, Zhang et al. (2010.Genes. 1: 85-101) teaches “One of the challenges in DNA methylation analysis is that although there is only one genome for each organism, there can be hundreds of epigenomes, because the DNA methylation changes with cell type and during development or disease processes and sometimes in response to environment. For example, in the human methylome cell type and developmental specific changes in the methylation pattern , changes in the ratio of non-CpG and CpG methylation or different methylation states of different gene copies in the same cell have been observed” (p. 87).
Hoglund et al (Nat Ecol Evol. 2020 Sep 21;4(12):1713–1724) teaches studied variations in methylation between domestic chickens and wild Red Junglefowl and the influence of methylation differences on gene expression (e.g., abstract and p. 3, second para). It is reported that there were significant differences in the methylomes and that the methylation changes appear to affect gene expression (p. 8). Hoglund (p. 8) states:
“By looking at inter-individual variation in methylation and how it correlates with gene expression, we find that around half of the correlations between DNA methylation and gene expression were positive, with the locations of these peak correlated windows being highly dispersed around the genes and not just limited to the promoter regions. This supports recent findings regarding the role of DNA methylation in gene expression, with DNA methylation being involved in increasing transcription factor binding activity 21 and also being present in enhancer regions, therefore outside promoter regions. Here we use inter-individual variation to show that such complex regulation is present through-out the genome, and can lead to both positive and negative correlations between DNA methylation and gene expression.”
Therefore, the findings regarding an association between methylation at LMRs and gut inflammation in broiler chickens cannot be predictably extrapolated to a representative number of additional avian species.
The specification does not provide any specific guidance as to how to predictably identify additional LMRs whose methylation level is indicative of gut inflammation or the level / severity of gut inflammation. There is also insufficient guidance provided as to how to extrapolate the findings obtained in the specification with broiler chickens to a representative number of alternative avian subjects.
Extensive experimentation would be required to identify additional LMRs whose methylation level is indicative of gut inflammation or the level / severity of gut inflammation and which can be used in combinations of two or three or five etc. to classify gut inflammation in broiler chickens and a representative number of other avian species. While methods for detecting LMRs and determining CpG methylation status are known in the art, such methods provide only the general guidelines that allow researchers to randomly determine the level of methylation of regions of the genome. The results of performing such methodology are highly unpredictable and thereby require undue experimentation.
Case law has established that '(t)o be enabling, the specification of a patent must teach those skilled in the art how to make and use the full scope of the claimed invention without ‘undue experimentation.'" In re Wright 990 F.2d 1557, 1561. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970) it was determined that '(t)he scope of the claims must bear a reasonable correlation to the scope of enablement provided by the specification to persons of ordinary skill in the art". The amount of guidance needed to enable the invention is related to the amount of knowledge in the art as well as the predictability in the art. Furthermore, the Court in Genetech Inc. v Novo Nordisk 42 USPQ2d 1001 held that '(I)t is the specification, not the knowledge of one skilled in the art that must supply the novel aspects of the invention in order to constitute adequate enablement".
In the present application, the claims do not bear a reasonable correlation to the scope of enablement for the reasons set forth above. In view of the unpredictability in the art, and the lack of disclosure in the specification and in the prior art, it would require undue experimentation for one of skill in the art to make and use the invention as broadly claimed.
11. The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Chen et al (Front. Vet. Sci. 2015. 3(14): 1-10) teaches assaying for biomarkers of gut barrier failure in chickens, which leads to gut inflammation (e.g., abstract and p. 4 “Gene Expression in Jejunal Mucosa by qRT–PCR”). It is stated that “any direct or
indirect damage on IEC may cause a breakdown in gut barrier and consequently, disruption of normal mucosal immune homeostasis that can potentially lead to uncontrolled chronic intestinal and systemic inflammation” (p. 2, col. 1). Chen teaches that the relative mRNA levels of genes that are possibly involved in gut barrier function and inflammation in jejunal mucosa of broiler chickens were determined and that relative levels of IL-8, IL1β, TGF-β4 and FABP6 increased while FABP2, occluding and MUC2 mRNA levels decreased (p. 4). However, Chen does not teach or suggest assaying for the level of methylation of LMRs in gut tissue samples from test chickens, relative to control samples from chickens that do not have gut inflammation, as indicative of gut inflammation in a test chicken or group of test chickens.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682
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