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 .
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Applicant has canceled all previously pending claims. Claims 8-13 have been added and are examined on the merits herein.
Response to Applicant’s Arguments and Amendments
The specification was objected to for including and referencing figures that were not placed in the drawings for the application. Applicant has removed these figures from the specification and placed them in the drawings, rendering this objection withdrawn. However, see new grounds of objection below.
Applicant has canceled all previous claims, and therefore all previous objections and rejections to the claims have been rendered moot. A complete response to Applicant’s claim amendments under the statutory requirements is provided below.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 112(a) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63/321,779, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Specifically, this application does not recite measuring phosphorylated TBK1 expression, nor does the application recite control thresholds. See also the new matter rejections in the 35 USC 112(a) section below. Therefore, this application will be given the effective filing date of the actual filing date of the instant application, 3/20/2023.
Specification
The amendment filed 2/4/2026 is objected to under 35 U.S.C. 132(a) because it introduces new matter into the disclosure. 35 U.S.C. 132(a) states that no amendment shall introduce new matter into the disclosure of the invention. The added material which is not supported by the original disclosure is as follows: Applicant has added paras. 8-13 which match in language to the newly added claims. However, as noted below in the 35 USC 112(a) Rejections, many of these limitations were not present in the original specification, and so constitute new matter. Applicant has also entirely rewritten the abstract of the instant specification to also include language from the newly amended claims that is not supported by the original specification, which also constitutes new matter.
Applicant is required to cancel the new matter in the reply to this Office Action.
Drawings
The drawings are objected to because Figures 2B, 3A, 5A, 5C, and 6A are not legible. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
The drawings are objected to as failing to comply with 37 CFR 1.84(p)(5) because they include the following reference character(s) not mentioned in the description: reference number 100 in Figure 1A. Corrected drawing sheets in compliance with 37 CFR 1.121(d), or amendment to the specification to add the reference character(s) in the description in compliance with 37 CFR 1.121(b) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 9 is objected to because of the following informality: “RNAseq / scRNAseq” should read “RNAseq/scRNAseq.” Appropriate correction is required.
Claim 10 is objected to because of the following informalities: the words “Debris Removal,” “Density,” and “Flow” should all begin with lowercase letters. Appropriate correction is required.
Claim 11 is objected to because of the following informalities: in line 1, “The method of claim 8, also comprising” should read “The method of claim 8, comprising.” Additionally, in line 2, it is recommended that “and wherein diagnosis requires” read “and wherein diagnosing the subject as having AD requires” to better match the language of claim 8. Appropriate correction is required.
Claim 12 is objected to because of the following informality: in line 2, “with p-value” should read “with a p-value.” Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 8-13 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.
It is noted that these new matter issues in the claims refer to the presence (or lack thereof) of the claimed limitations in the original disclosure, including the specification, drawings, and claims, and not in Applicant’s newly amended specification. See MPEP 608.04. It is noted that new matter has been considered with regard to the guidance on inherent function, theory, or advantage presented in MPEP 2163.07(a).
Claim 8 recites the measurement of expression levels in particular samples, comparing the expression levels to those of controls, diagnosing the subject as having AD if thresholds are exceeded. The claim specifically recites a human subject, and the original disclosure appears to only isolate microglia from mouse subjects (e.g. para. 39 and Figure 4 of the original disclosure). While the original disclosure also notes the use of TPK1 in the STING signaling pathway generally (e.g. Figure 2), phosphorylated TBK1 is not discussed in any capacity. The original disclosure is silent as to the use of any control thresholds or the use of age-matched individuals, though controls are generally recited, which are presumed to be from non-AD individuals (see Figure 7 for example). These aspects of the claim are therefore considered new matter.
Claims 9-12 are rejected due to their dependence on rejected claim 8.
Claim 10 recites methods for the isolation of microglia and leukocytes. None of the methods described for isolated microglia are recited by the original disclosure, and so are considered new matter.
Claim 11 recites the measuring of phosphorylated TBK1 protein via ELISA, and the use of both elevated ISG mRNA and phospho-TBK1 for diagnosis of AD. ELISA is taught in para. 11 of the original disclosure as a general way of monitoring STING signaling activation, but its specific use for measuring p-TBK1 is not recited. While TBK1 is also shown to be elevated in AD mouse models (Figure 4), phosphorylated TBK1 is not discussed in any capacity, much less for diagnosis, and so this is considered new matter.
Claim 12 recites a diagnostic threshold for each target, but this threshold is not recited in Applicant’s original disclosure, and so is considered new matter.
Claim 13 recites primers and probes, positive and negative control materials, and instructions for use that recite control thresholds for said genes. The original disclosure is silent as to: primers and probes of any kind, positive or negative controls, instructions of any kind, and control thresholds. The specification does recite qRT-PCR (see Figures 3B and 5B and current paras. 38 and 40 of the instant specification), which is considered to inherently involve primers and probes. However, the claim requires primers and probes for qRT-PCR of IRF7, IFIT3, OASL1, and MX1, but in the described figures, only IRF7 is shown as undergoing qRT-PCR. Expression of IFIT3, OASL1, and MX1 are all shown in Figure 7, but the specific test performed in Figure 7 is not recited in the original disclosure. The specification also discusses controls generally (e.g. see Figures 4 and 7), but no thresholds are recited. Therefore, this claim is considered to contain new matter with regard to the entirety of (ii) and (iii), and with regard to primers and probes for qRT-PCR for IFIT3, OASL1, and MX1.
Based on Applicant’s lack of detail in the specification regarding the claimed invention and the scope of the new matter rejections, it is not clear that Applicant had possession of the claimed invention at the time of filing, and so the claims are rejected for failing to comply with the written description requirement.
Claim Rejections - 35 USC § 112(b)
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 8-13 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.
Regarding claim 8, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). In this specific instance, it is unclear if the specifically stated ISG mRNAs (IRF7, IFIT3, OASL1, and MX1) must be measured in the claim, and if so, how many must be measured. The claim will be interpreted to mean that at least two ISG mRNA levels must be measured, and that the ISG mRNAs can be any ISG mRNAs, and may or may not include those listed in the “such as” phrase.
Claim 8 is also rejected because in line 3, the phrase “defined ISG mRNA levels” are recited. It is unclear what would make a particular ISG mRNA level “defined” verses “not defined,” and this is not made clear in the instant specification.
Claim 8 is also rejected because the phrasing “measuring phosphorylated TBK1 and defined ISG mRNA levels” generally renders the scope of the method unclear. Specifically, it is unclear whether the language of the claim indicates that TBK1 mRNA is intended to be measured, in which case it is unclear how phosphorylated TBK1 expression specifically could be measured, as mRNAs are not typically phosphorylated, or if any phosphorylated TBK1 measurements would suffice. Given that phosphorylation is typically measured in the context of proteins, it will be interpreted as though protein expression of phosphorylated TBK1 is to be measured.
Claim 8 is also rejected because in line 4, “the measured expression levels” lacks sufficient antecedent basis, as “measured expression levels” are not generally recited earlier in the claim. It will be interpreted as though these expression levels refer to those of any of the markers recited in the previous step of the method. If Applicant wishes for this phrase to include expression of all of the markers recited earlier in the claim, it is recommended to establish these as expression levels, by stating “measuring phosphorylated TBK1 and ISG mRNA levels to generate measured expression levels,” or similar phrasing.
Claims 9-12 are rejected due to their dependence on rejected claim 12.
Claim 9 is also rejected because it appears to be referring to methods of measuring mRNA, but the full scope of this measuring is unclear in light of the indefiniteness associated with the measuring of the phosphorylated-TBK1 described above. The claim will be interpreted as though the PCR/sequencing methods are referring to the measuring of the ISG mRNA only.
Claim 10 contains the trademarks/trade names Accutase and Ficoll. Where a 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 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See 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 trademarks/trade names are used to identify/describe microglia and leukocyte isolation methods, respectively, and, accordingly, the identification/description is indefinite.
Claim 10 is also rejected because it describes isolation methods for microglia and leukocytes, where one of each isolation method must be used, as evidenced by the language “and the leukocytes” in line 3. However, in claim 8, from which this claim depends, either microglia or leukocytes may be used, and it is never stated that both must be used. This renders the isolation methods of claim 10 unclear in view of the method of claim 8. The claim will be interpreted as though either microglia or leukocyte isolation may be performed.
Claim 12 is also rejected because the phrase “the diagnostic threshold” lacks sufficient antecedent basis, as this phrase is not used in claim 8, from which this claim depends. Additionally, this claim recites each measured “target” but no targets are recited in claim 8, and so this phrase also lacks antecedent basis. It is recommended to amend “the diagnostic threshold” to be directed to the control thresholds and to amend the “target” language to instead be focused on the expression levels measured in claim 8. The claim will be interpreted as though it is referring to these aspects of claim 8, respectively.
Claim 13 is rejected because in (iii), control thresholds for “said genes” is recited, but “genes” are not generally recited earlier in the claim. Therefore, this phrase lacks significant antecedent basis. It will be interpreted as though the genes are the same as those recited in (i) of the claim.
Claim Rejections - 35 USC § 101
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 8-12 are rejected under 35 U.S.C. 101 because the claimed invention is directed to judicial exceptions without significantly more. The claims recite a natural law and abstract ideas.
Claim 8 is directed to a method for diagnosing Alzheimer’s disease via measuring expression levels The natural law recited is the relationship between the expression level of the biomarkers and the presence of AD in a subject. The abstract ideas are the comparing and diagnosing steps, as these steps simply involve comparing expression levels to one another, and thus can be done in the human mind. The judicial exceptions are not integrated into a practical application because there is no required active treatment step or other step that integrates the judicial exceptions into a practical application. See MPEP 2106.04(d)(2). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exceptions because they do not amount to more than well-understood, routine, and conventional activity in view of Yu et al. (US 2023/0149419 A11, hereafter “Yu”). Yu teaches methods for treating neuroinflammatory disease (Abstract). The reference specifically mentions Alzheimer’s disease, and notes the importance of determining diagnostic and treatment methods for said disease (paras. 3-4). Yu teaches measuring mRNA expression levels of genes associated with Alzheimer’s disease in microglia from control and AD mouse models (e.g. paras. 64, 111, 121-122, and 124). Protein expression in microglia was also examined (e.g. paras. 123-124 and 127-128).
Thus, the claim as a whole is directed to judicial exceptions without significantly more.
Claim 9 depends on claim 8 and defines methods that must be used for measuring mRNA levels. The claim does not integrate the judicial exceptions into a practical application because there is no required active treatment step or other step that integrates the judicial exceptions into a practical application. See MPEP 2106.04(d)(2). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exceptions because they do not amount to more than well-understood, routine, and conventional activity in view of Yu, which teaches qRT-PCR (e.g. paras. 103 and 109).
Thus, claim 9 is directed to judicial exceptions without significantly more for the same reasons described above for claim 8.
Claim 10 depends on claim 8 and defines methods for isolating cells. The claim does not integrate the judicial exceptions into a practical application because there is no required active treatment step or other step that integrates the judicial exceptions into a practical application. See MPEP 2106.04(d)(2). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exceptions because they do not amount to more than well-understood, routine, and conventional activity in view of Bordt et al. (STAR Protocols, 2020, hereafter “Bordt”). Bordt teaches methods for isolating microglia, specifically involving isolating CD11b+ cells using magnetic bead columns (Summary). This method also utilizes Debris Removal Solution (Figure 2) to remove myelin or other debris and further isolate microglia (pages 4-5, joining para.). After magnetic bead isolation, flow cytometry was also used for sorting cells (page 11, para. 3).
Thus, claim 10 is directed to judicial exceptions without significantly more for the same reasons described above for claim 8.
Claim 11 depends on claim 8 and requires a particular method for measuring p-TBK1. The claim also recites diagnostic criteria, but this only serves to further the judicial exceptions of claim 8. The claim does not integrate the judicial exceptions into a practical application because there is no required active treatment step or other step that integrates the judicial exceptions into a practical application. See MPEP 2106.04(d)(2). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exceptions because they do not amount to more than well-understood, routine, and conventional activity in view of Yu, which teaches the use of ELISA to quantitatively analyze particular proteins (e.g. paras. 78 and 83), and specifically utilizes this method for analyzing proteins from microglia (e.g. paras. 101-102, 108, 115, and 127).
Thus, claim 11 is directed to judicial exceptions without significantly more for the same reasons described above for claim 8.
Claim 12 depends on claim 8 and requires particular diagnostic criteria. This only serves to further the judicial exceptions of claim 8. The claim does not integrate the judicial exceptions into a practical application because there is no required active treatment step or other step that integrates the judicial exceptions into a practical application. See MPEP 2106.04(d)(2). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exceptions because they do not amount to more than well-understood, routine, and conventional activity.
Thus, claim 12 is directed to judicial exceptions without significantly more for the same reasons described above for claim 8.
Claim 13 is rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception without significantly more. The claim recites natural products.
Claim 13 is drawn to a diagnostic kit comprising primers and probes, control materials, and instructions. The instructions are considered nonfunctional descriptive material and are not given patentable weight, as it is printed matter that does not inform the structure of the primers, probes, or control materials. See MPEP 2111.05. The “control materials” are also not defined by the instant specification, and so can contain solely natural products, such as water for reactions. Regarding the primers and probes, though it is not clear that oligonucleotides having the features recited in claim 13 exist in nature, the claimed oligonucleotides are, nevertheless, judicial exceptions because they are derived from naturally occurring molecules and are not required to possess any structural or functional differences relative to their naturally occurring counterparts. For example, the oligonucleotides of claim 13 are not required to include a detectable label (it is noted that while the primers and probes are “for qRT-PCR,” and so the probes may potentially be intended to have detectable labels, this structure is not recited in the claim). As well, the oligonucleotides do not have a function that differs from that of the naturally occurring counterparts since, like the naturally occurring counterparts, the claimed oligonucleotides hybridize to specific gene targets. See also MPEP 2106.04(c) II C 2, which specifically states that primers are not markedly different from natural products.
Thus, the claim as a whole is directed to a group of products that are not markedly different from their naturally occurring counterparts.
Claim Interpretation
Regarding the “control thresholds” of claim 8, as noted above in the 35 USC 112(a) Rejections section, this phrase is not recited in the instant specification. It will be interpreted as though any prior art that compares control expression levels to expression levels in patients/samples with AD or at risk of AD will be considered to meet the comparing limitation of the claim, as a control threshold can simply be a control level of expression.
Regarding the instructions recited in (iii) of claim 13, as noted above in the 35 USC 101 Rejection, the instructions are considered nonfunctional descriptive material and are not given patentable weight. In interpreting this in light of the prior art, said prior art will be considered to read on this limitations if it generally teaches instructions, with no limit to the content of said instructions.
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.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(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.
Claim 13 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Gajewski et al. (US 2020/0010557 A1, hereafter “Gajewski”).
Gajewski teaches the detection of expression of particular biomarkers (Abstract). The markers can be those that are differentially expressed on the surface of T-cells or cell surface receptors, or those that correlate with 4-1BB and LAG-3 expression (para. 6). These markers are in part listed in Table 2 (para. 12). The reference specifically teaches the use of kits for the detection methods of the invention, where said kits can have probes and amplification oligonucleotides (e.g. primers), positive and negative control samples, and instructions (para. 96). Table 2 provides a list of differentially regulated genes, and includes IRF7, IFIT3, OASL1, and MX1. Para. 102 of the reference provides evidence that the primers and probes of the invention can be used for qRT-PCR.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Romagnoli et al. (Int. J. Mol. Sci., 2020, hereafter “Romagnoli”), in view of Abreha et al. (J. Biol. Chem., 2021, hereafter “Abreha”), in view of Zhang et al. (Neuropsychiatric Disease and Treatment, 2020, hereafter “Zhang”), and in view of Yu et al. (US 2023/0149419 A12, hereafter “Yu”).
Romagnoli teaches the measurement of several mRNAs in relation to Alzheimer’s disease (AD), including IRF7 (Abstract). mRNA was measured utilizing quantitative reverse-transcription PCR (page 10, “Quantitative Reverse-Transcription Polymerase Chain Reaction”; instant claim 9), where the RNA was taken from frozen hippocampus and temporal cortex samples from AD patients and controls (page 9, “Post-Morten Human Brain Tissue” and page 10, “RNA Isolation”). Figure 1 shows the mRNA expression results for both sample types, where, for IRF7, there was a significant decrease in expression in both brain regions for a group of AD patients. However, there was also a non-significant increase in expression for a group of AD patients. Regarding this disparity, Romagnoli states, “Differential expression patterns might represent different AD clinical stages,” (page 8, para. 4).
Abreha teaches the examination of TBK1 activation in relation to Alzheimer’s disease (Abstract and page 2, column 1, para. 2). TBK1 generally shows a significant 5.32 log-fold upregulation in AD samples compared to controls (Figure 1B, page 2, column 1, para. 1). Figure 2B shows a comparison of phospho-TBK1 levels in AD samples and controls generated from Western blotting, and a similar statistically significant increase was seen, where p-TBK1 levels were nearly doubled in AD samples (see figure caption and page 3, column 1, para. 1). These analyses utilized human brain tissues (page 9, column 2, para. 3). Abreha does described performing qPCR, but this is done for expression levels related to transgenic flies (see Figure 6C and page 11, “Quantitative real-time PCR (qRT-PCR)”).
Zhang teaches the measurement of several proteins and mRNAs associated with AD in transgenic mice, including OASL1 (Abstract). Both qRT-PCR and Western blots were used (see page 2171). Figure 6 shows that compared to the control group, expression of OASL1 mRNA is significantly decreased in the AD group. This trend does not appear as strongly when observing OASL1 protein expression, as a decrease is shown in the AD group compared to the controls, but this is not significant.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to add the biomarkers described by Abreha and Zhang into the method of Romagnoli. Abreha and Zhang describe additional biomarkers that exhibit significant differences in AD patients versus controls, and so by utilizing these additional biomarkers, further diagnostic criteria for AD can be generated, and additional disease mechanisms can be uncovered, which can pave the way for better detailing of disease onset and uncovering potential treatment methods. By incorporating additional biomarkers into the method of Romagnoli that already have known trends, this can also increase the accuracy of the method, as Romagnoli notes that groups of AD patients appear to show different expression level trends for particular biomarkers, and so these markers, if used alone, may confuse potential diagnostics and further research. There would be a reasonable expectation of success in utilizing these biomarkers as their expression is already successfully measured in Abreha and Zhang.
Additionally, as shown by Zhang, protein and mRNA expression levels for a single gene do not necessarily perfectly correlate. Thus, it would additionally be prima facie obvious to maintain the mRNA and protein expression measurements of Abreha and Zhang specifically in the method of Romagnoli, in view of Abreha, and in view of Zhang. This would ensure that the known expression relationships between control and AD groups are maintained, further bolstering the advantages described in the paragraph above.
Furthermore, each of these references compares expression levels in samples for patients already known to have AD with those of controls. However, the ordinary artisan would recognize that the expression values established by these references could be used as points of comparison for future analysis in order to diagnose a patient with AD. This is generally the purpose of discovering useful biomarkers, and it would allow for patients to be definitively diagnosed, particularly in early stages of disease, allowing for earlier interventions and potentially improving patients’ quality of life and slowing disease progression. There would be a reasonable expectation of success in using the combination of references in this manner as it would simply involve performing the method, with the same general steps and analyses, on new samples.
However, none of these references teach utilizing human microglia or leukocyte cells.
Yu teaches methods for treating neuroinflammatory disease (Abstract). The reference specifically mentions Alzheimer’s disease, and notes the importance of determining diagnostic and treatment methods for said disease (paras. 3-4). Yu teaches measuring mRNA expression levels of genes associated with Alzheimer’s disease in microglia from control and AD mouse models (e.g. paras. 64, 111, 121-122, and 124). Protein expression in microglia was also examined (e.g. paras. 123-124 and 127-128). mRNA expression levels in microglia are specifically noted to be assessed with qRT-PCR (e.g. paras. 103, 109, 120, 127, and 136). The reference specifically teaches that microglia is implicated in neuroinflammation, and that the activation of microglia is “considered to be an important mechanism for progression of neurodegenerative disease as important pathological markers,” (para. 2).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to utilize microglia cells to measure the mRNA and protein expression levels of Romagnoli, in view of Abreha, and in view of Zhang. Yu teaches the importance of microglia to the development of Alzheimer’s, as well as generally describes the importance of better understanding Alzheimer’s disease. The reference also shows the successful use of microglia to determine mRNA and protein expression levels. Thus, this change would amount to a simple substitution of the sample type of Romagnoli, in view of Abreha, and in view of Zhang for that of Yu. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.“ As noted above, Yu teaches that mRNA and protein expression can be assessed via microglia cells, and so this change would result in the predictable determination of expression levels of the genes of Romagnoli, in view of Abreha, and in view of Zhang.
Thus, claims 8-9 are prima facie obvious over Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Romagnoli et al. (Int. J. Mol. Sci., 2020, hereafter “Romagnoli”), in view of Abreha et al. (J. Biol. Chem., 2021, hereafter “Abreha”), in view of Zhang et al. (Neuropsychiatric Disease and Treatment, 2020, hereafter “Zhang”), in view of Yu et al. (US 2023/0149419 A1, hereafter “Yu”), and further in view of Bordt et al. (STAR Protocols, 2020, hereafter “Bordt”).
Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu teach the methods of claims 8-9, as described above. However, none of the references teach the microglia isolation methods utilized in the instant claim.
Bordt teaches methods for isolating microglia, specifically involving isolating CD11b+ cells using magnetic bead columns (Summary). This method also utilizes Debris Removal Solution (Figure 2) to remove myelin or other debris and further isolate microglia (pages 4-5, joining para.). After magnetic bead isolation, flow cytometry was also used for sorting cells (page 11, para. 3). Bordt teaches that this method can be used to isolate cells at different developmental ages (page 11, para. 4), and the method can be used on fresh frozen human brain tissue (pages 11-12, joining para.). This method also appears to allow CD11b+ cells to be successfully cultured over a longer time period than is typical for this cell type (page 12, para. 2).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to utilize the microglia isolation method of Bordt in the method of Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu. In Yu, microglia cells were isolated via culturing brain tissue in particular reagents and in vitro tapping (paras. 100 and 126), at least for some of the working examples. Bordt provides several benefits of their method that would motivate the ordinary artisan to choose it over other isolation methods – specifically, more time for culturing, thereby potentially generating more cells for analysis, and the ability to specifically sort cells by age, which would be particularly useful in AD applications, to see how cells change as the disease progresses. As the method of Bordt is also specifically drawn to isolating microglia cells, and teaches that their methods can be used on human samples, there would be a reasonable expectation of success. This change in isolation methods would also amount to a substitution of the isolation methods of Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu for those of Bordt. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.“ As both methods result in successful microglia isolation, this substitution would yield predictable results.
Thus, claim 10 is prima facie obvious over Romagnoli, in view of Abreha, in view of Zhang, in view of Yu, and further in view of Bordt.
Claim 11-12 are rejected under 35 U.S.C. 103 as being unpatentable over Romagnoli et al. (Int. J. Mol. Sci., 2020, hereafter “Romagnoli”), in view of Abreha et al. (J. Biol. Chem., 2021, hereafter “Abreha”), in view of Zhang et al. (Neuropsychiatric Disease and Treatment, 2020, hereafter “Zhang”), in view of Yu et al. (US 2023/0149419 A1, hereafter “Yu”), and further in view of Frigerio et al. (Cell Reports, 2019, hereafter “Frigerio”).
Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu teach the methods of claims 8-9, as described above.
Regarding claim 11, Yu teaches the use of ELISA to quantitatively analyze particular proteins (e.g. paras. 78 and 83), and specifically utilizes this method for analyzing proteins from microglia (e.g. paras. 101-102, 108, 115, and 127). The phosphorylated TBK1 in Abreha is measured utilizing Western blotting (see Figure 2, for example). However, it would be prima facie obvious to substitute the protein expression methodology of Abreha with that of Yu in the overall method of Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.“ Both Western blotting and ELISA are known ways of measuring protein expression, as evidenced by Abreha and Yu, respectively, and Yu teaches that ELISA methods are compatible with microglia samples. Thus, this substitution would produce the predictable result of successful p-TBK1 protein expression measurements.
Regarding AD diagnosis, p-TBK1 expression is already shown by Abreha to be elevated in AD compared to controls, being nearly 2x higher in AD patients compared to controls with a p < 0.001 (Figure 2B). Romagnoli also teaches that IFN-α expression is significantly elevated in some AD patients in the hippocampus data, with a p < 0.01, but expression was also decreased in some AD patients.
Thus, it is not clear from the specific combination of Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu described above that upregulation of ISG mRNA with specific diagnostic criteria could be used to diagnose AD.
Frigerio teaches gene expression profiles of more than 10,000 microglial cells (Summary). Figure 1B notes different gene clusters in microglia, where the IRM cluster is enriched for interferon genes (see figure caption). This cluster shows high expression of several genes, including IFIT3 and IRF7 (Figure 1D and page 1297, column 1, para. 2). The reference compared mice with AD mutations (APP mice) to wild-type controls (C57BL/6J; see Figure 1), and found that the IRM cluster had higher expression in APP mice as these mice aged, and had higher expression compared to that of controls at nearly every age (Figure 1C). An IRM state is also shown as more common in APP mice compared to controls in Figures 2F-G. Figure 3A shows the significant differences in gene expressions for the different clusters, with IRM showing some significant differences at all p-values. Figures 3B-C also show measurements of up- and downregulated genes in altered microglia compared to homeostatic microglia. Figures 1D and 5B also show normalized gene expression for an array of chosen genes, and for the IRM genes IRF7 and IFIT3, which show expression orders of magnitude higher in IRM clusters compared to other clusters.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Frigerio in the method of Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu to arrive at the methods of instant claims 11-12. Specifically, Frigerio teaches the analysis of thousands of genes potentially associated with AD-risk in microglia. The reference also teaches different clusters of genes and their implications in abnormal microglia cells, as well as expression and p-value analysis for the different genes/clusters. Generally, Frigerio showed that the IRM genes are associated with a higher expression level in AD-mice compared to controls. Taking this information in light of the teachings of Romagnoli, in view of Abreha, in view of Zhang, and in view of Yu, and particularly in view of Abreha, which teaches increased p-TBK1 levels in AD patients, the ordinary artisan would be motivated to develop an assay that looks for overall upregulation of expression for mRNA and p-TBK1 as a diagnostic criteria for AD. As the IRM genes of Frigerio (such as IFIT3 and IRF7) show upregulation associated with AD, the ordinary artisan could examine expression in all of these genes and then observe the overall trend to determine if a particular subject has AD. This would be a simpler assay analysis than analyzing individual genes that may have different relationships to AD (i.e. up- or downregulation) and would allow for a more robust analysis, as this analysis would be less prone to noise or error in determining the expression levels for individual genes. Additionally, Frigerio notes that for many of the IRM genes, expression greatly increased in IRM clusters compared to the expression of these genes in non-IRM clusters. The reference also details genes with p-values of 0.01 and lower for particular expression analyses. Thus, the ordinary artisan would additionally be motivated to choose genes in the overall assay of Romagnoli, in view of Abreha, in view of Zhang, in view of Yu, and further in view of Frigerio that display the strongest differences in expression in AD compared to that of controls – this would ensure that any statistical analyses are powerful enough to act as accurate diagnostics. The ordinary artisan could easily arrive at the diagnostic criteria of claim 12, and would likely choose even higher expression and p-value thresholds, given those shown by Frigerio.
Thus, claims 11-12 are prima facie obvious over Romagnoli, in view of Abreha, in view of Zhang, in view of Yu, and further in view of Frigerio.
Conclusion
No claims are currently allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/F.F.G./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
1 It is noted that this reference is a continuation of PCT/KR21/15855, which was filed no November 4, 2021, before the effective filing date of the claimed invention. This PCT application was submitted in the Korean language, so for ease of prosecution, the US application is cited and referenced herein.
2 It is noted that this reference is a continuation of PCT/KR21/15855, which was filed no November 4, 2021, before the effective filing date of the claimed invention. This PCT application was submitted in the Korean language, so for ease of prosecution, the US application is cited and referenced herein.