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. Claim 3 has been canceled. Claims 8-14 remain withdrawn. Claims 1-2 and 4-7 are pending and are examined on their merits herein.
Response to Applicant’s Amendments
Drawing Objections
The drawings were objected to because Figures 11-13 were not legible and Figure 14 appeared to be cutoff. In light of Applicant’s replacement drawings submitted 2/25/2026, these objections have been withdrawn.
Claim Objections
Claims 1-4 and 6-7 were objected to due to various informalities. These objections have been withdrawn for all currently pending claims. Claim 3 has been canceled, and so this objection has been rendered moot. See also new grounds of objection below.
35 USC 112(b) Rejections
Claims 1-7 were rejected due to various indefiniteness issues. The rejections for claims 1-2 and 6-7 have been withdrawn. Claim 3 has been canceled, and so this rejection has been rendered moot. The rejections for claims 4-5 have been maintained-in-part. See the 35 USC 112(b) rejections below.
35 USC 112(d) Rejections
Claim 2 was rejected for being of improper dependent form. In light of Applicant’s amendments to the claims submitted 2/25/2026, this rejection has been withdrawn.
35 USC 103 Rejections
Claims 1-5 and 7 were rejected under 35 U.S.C. 103 as being unpatentable over Cutliffe et al. (US 2015/0259728 A1), in view of Stämmler et al. (Microbiome, 2016), and in view of Mitter et al. (US 2016/0330976 A1).
Claim 6 was rejected under 35 U.S.C. 103 as being unpatentable over Cutliffe et al. (US 2015/0259728 A1), in view of Stämmler et al. (Microbiome, 2016), in view of Mitter et al. (US 2016/0330976 A1), and further in view of Sung et al. (WO 2021/102293 A1).
Applicant’s arguments and amendments have been thoroughly reviewed and considered. These rejections have been maintained for all currently pending claims. Claim 3 has been canceled, and so this rejection has been rendered moot. See “Response to Applicant’s Arguments” below.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that Cutliffe does not teach the use of spike-in sequences (Remarks, page 17). It is noted that the Examiner agrees with this assessment, and states as such in para. 31 of the Non-Final Rejection (“However, Cutliffe does not teach the addition of spike-in sequences before DNA extraction or specifically counting cell numbers.”). Applicant states that this lack of teaching means that there can be no finding of obviousness, but this ignores the additional references recited in the rejection of claim 1 (Stämmler and Mitter) and amounts to a piecemeal analysis of the references.
In response to Applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant states that Stämmler, which is used in the Non-Final Rejection to teach spike-in sequences, is directed to 16S rRNA sequencing only, and so Applicant states that “The Office Action seems to allege that the skilled person would switch between 16S rRNA sequencing and shotgun metagenome sequencing without any inventive skill,” (Remarks, page 18). Applicant also argues that Stämmler only teaches calibrations of ratios of absolute abundances and not absolute quantification.
In the instant claims, the spike-in sequences are placed into a sample, all nucleic acids are extracted from the sample, the nucleic acids are sequenced, and then data analysis occurs. Specifically in step (d) of claim 1, the use of relative abundances or copy numbers is recited. This is then used in step (f) to determine cell number information. No precise absolute quantification in step (d) is required. In Stämmler, the absolute abundance of the spike-in bacteria is known (page 2, column 1, para. 3 and page 2, column 2, para. 2), and this known absolute abundance is then used to create ratios of absolute abundance for the bacteria of interest within a sample (e.g. Figures 5b and c). These ratios were used to show if there was an increase or decrease in target bacteria over time (such as after a treatment, as shown in Figure 5c). In combining the teachings of Stämmler with those of Cutliffe, it is noted that the latter reference does specifically teach genomic shotgun sequencing of microbial species. Even though the spike-ins of Stämmler are not taught to be sequenced with shotgun sequencing, the ordinary artisan would still recognize that these sequences could be sequenced with shotgun sequencing, along with all of the other microbial microbiome species taught by Cutliffe. Additionally, this combination is stated to “involve adding multiple spike-ins to a sample before extraction occurred, finding copy numbers and relative abundances, and then using these values to determine absolute abundance, as is done in Stämmler,” (see para. 33 of the Non-Final Rejection). Even though these are ratios of absolute abundance, does not change how the combination of references reads on the instant claim (which does not require absolute abundance information). Furthermore, the Examiner states in para. 34 of the Non-Final Rejection that this combination, and Stämmler in particular, does not teach finding cell numbers for their species of interest. This is then taught by the Mitter reference (see paras. 35-36 of the Non-Final Rejection). In combining Cutliffe, in view of Stämmler, and in view of Mitter, a substitution of the absolute quantification methods of Stämmler with the determination of the number of DNA copies in a sample as shown in Mitter is made. Applicant does not substantively address this combination in their Remarks.
In relation to the amended claims, it is noted that the portion of Mitter cited in the Non-Final Rejection, paras. 460-461, teach determining the number of DNA genomic copies in a sample (which would be analogous to cell number in the instant claims) using the amount of DNA present and the length of the genome of the species of interest. To obtain the information for this calculation, calibration curves were created for the species of interest (with an unknown number of copies) and species with known concentrations (para. 461).
In considering this combination of references in view of the instant claims, Cutliffe teaches step (a), step (c), the shotgun sequencing and species identification of step (d), and use of microbiome information to relate to health conditions in step (g). In combining Cutliffe in view of Stämmler, the references additionally teach step (b) and the calculation of copy numbers and relative abundances in step (d). In combining Cutliffe, in view of Stämmler, and in view of Mitter, the references additionally teach steps (e) and (f).
Thus, this combination of references teaches each of the limitations of newly amended claim 1.
On page 19 of their Remarks, Applicant also states various limitations that the prior art does not allegedly teach all of. These limitations are considered to include items that, while potentially encompassed by claims 1 and/or 2, do not read one-for-one onto the limitations of these claims - these includes precise x- and y-axis mapping for calibration curves and use of linear regression. Outside of these narrower than claimed limitations, as the Examiner noted in the paragraph above, the cited combination of references is believed to teach each limitation of instant claims 1-2.
Thus, Applicant’s arguments are not considered persuasive, and the prior art rejections presented in the Non-Final Rejection have been maintained for all currently pending claims. Additional details as to the teachings of each reference and how they relate to the claimed limitations have been provided below in the 35 USC 103 rejections for clarity of the record. As claims 1 and 2 have been amended, the wording of the rejections has also been slightly altered to ensure that each limitation is clearly linked to the references. As the basic thrust of the rejections has not been changed with this altered wording, this is not considered to be new grounds of rejection.
Claim Objections
Claim 1 is objected to because of the following informalities:
in lines 1-2, “of a human mammal” should read “of a human or mammal” based on the wording used later in the claim.
In step b, line 2 reads “standards comprises” but should read “standards comprise.”
The period after “step d” in line 2 of step e should be removed.
In step g, line 1 reads “each microbial species or strains” but should read “each microbial species or strain.”
Appropriate correction is required.
Claim 6 is objected to because of the following informality: in lines 3-4, “microbial species and strains” should read “microbial species or strains.” Appropriate correction is required.
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 4-5 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.
Claim 4 is rejected because it is overall unclear. The claim is drawn to the impact of additional variables on cell numbers, but does not recite a step of actually measuring or noting qualities of an additional variable in the method of claim 1. Lines 4-5 of the claim state that this is done via “combining and organizing the cell number data from step f with the mapping data obtained from step g,” but this does not cite measuring any additional variables or determining the impact of them.
Claim 5 is also rejected due to its dependence on rejected claim 4.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4-5, and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Cutliffe et al. (US 2015/0259728 A1), in view of Stämmler et al. (Microbiome, 2016), and in view of Mitter et al. (US 2016/0330976 A1).
Cutliffe teaches methods for profiling a microbiome and methods for assessing or predicting health status in a subject (Abstract). The subject examined can be a human that may be asymptomatic or have symptoms of a disease (para. 25). The biological sample used can be indicative of a gut microbiome (para. 22). The reference teaches microbiome profiling involving obtaining genetic information for a large number of microbes via sequencing methods (para. 12). Entire genomes may be analyzed when creating a microbiome profile (para. 70). Microbiome profiles can be used in diagnostic assays, where a particular combination of microbes found in combination with other characteristics such as age, weight, gender, and medical history, and risk factors can be used to generate a score that can predict disease status (para. 76; instant claims 4-5). The disease examined can be IBD or a number of other diseases (para. 21). Cutliffe teaches that whole genome shotgun sequencing can be used to determine microbiome profiles and distributions (para. 92). Cutliffe also notes that their microbe identification can be at the strain or sub-strain level (e.g. paras. 10 and 33-35) and notes that shotgun sequencing is compatible with strain identification (para. 108). The reference also specifically states that a microbiome profile can quantify the microbes in the microbiome (para. 65), and that such quantification can be incredibly accurate (para. 145).
Thus, in relation to instant claim 1, Cutliffe teaches step (a), step (c), the shotgun sequencing and species identification of step (d), and the use of microbiome information to relate to health conditions of step (g).
However, Cutliffe does not teach the addition of spike-in sequences before DNA extraction or specifically counting cell numbers. The reference does note that known quantities of non-commensal strains of bacteria can be added to a sample to aid in developing microbial profiles (para. 91), but this appears to be more related to measuring background variation and calibrating sequencing (paras. 34 and 135).
Stämmler teaches the spiking of exogenous bacteria into samples to obtain absolute abundances of endogenous bacteria in samples of patients undergoing allogeneic stem cell transplantation (Abstract). Three spike-in bacteria were chosen, Salinibacter ruber, Rhizobium radiobacter, and Alicyclobacillus acidiphilus, none of which are found in humans. Whole bacteria were spiked-in at fixed, known amounts (page 2, “Choice of spike-in bacteria”). The bacteria were spiked into collected human stool samples and DNA extraction was performed (page 9, “Human ASCT specimens” and “Mouse specimens,” note that it is stated that spiking for human samples was performed as it was for mouse samples, where spiking occurred before extraction). Metagenomic DNA was then extracted and pyrosequencing was done (pages 9-10, “Amplification of V3-V6 16S rDNA variable region and 454 pyrosequencing” and Figure 6). Quantification of copy numbers of total bacteria and spike-ins were determined, and these were used to obtain relative abundances for operational taxonomic units (OTUs) in the samples, and absolute numbers of OTUs were then found (pages 10-11). Stämmler teaches that the use of spike-in standards allows for sensitivity to microbial loads within a sample, allows for concentrations to be standardized across different samples (page 2, column 1, para. 2), and provides more controlled and accurate results, so much so that the reference recommends that spike-ins become standard routine when analyzing microbiomes (page 8, “Conclusion”).
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 spike-in methods taught by Stämmler in the method of Cutliffe. Specifically, this would involve adding multiple spike-ins to a sample before extraction occurred, finding copy numbers and relative abundances, and then using these values to determine absolute abundance, as is done in Stämmler. Stämmler teaches many advantages of spike-ins that would be motivating to the ordinary artisan, and shows that spike-in methods are compatible with microbiome analyses, so much so that they argue such methods should become standard practice. This would also provide the ordinary artisan with a reasonable expectation of success.
Thus, the combination of Cutliffe in view of Stämmler additionally teaches step (b) and the calculation of copy numbers and relative abundances in step (d) in relation to instant claim 1.
However, Stämmler does not specifically teach finding cell numbers for their species of interest, as they mainly deal with abundances of sequence reads and ratios between spike-ins and endogenous sample bacteria (Figure 5 and pages 6-8, “Calibration to microbial loads reveals absolute increase of Enterococcus in the intestine during allogeneic stem cell transplantation”).
Mitter teaches methods related to libraries of microorganisms (Abstract). In Experiment 2, to determine the number of DNA copies in a sample, the quantity of DNA divided by the fragment length and average weight of a base pair was used in combination with Avogadro’s number (see the equation between paras. 460-461). These paragraphs also teach determining the number of DNA genomic copies in a sample (which would be analogous to cell number in the instant claims) using the amount of DNA present and the length of the genome of the species of interest. To obtain the information for this calculation, calibration curves were created for the species of interest (with the unknown number of copies) and species with known concentrations.
The method of Cutliffe in view of Stämmler already involves copy number determinations of total and spike-in bacteria. However, the ordinary artisan would recognize the use for finding the specific number of copies for each microbial organism in a sample (including those of the endogenous bacteria), as these would be more accurate measurements than measuring sequencing reads (where biases may be introduced) or ratio values, as the cell copy number would better represent true abundance in a sample. This would then be a substitution of the absolute quantification method of Cutliffe in view of Stämmler for that of Mitter, including the use of the calibration curves to determine copies of target sample sequences relative to known sequence concentrations. 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.” Using the equation of Mitter would yield the predictable results of determining the number of DNA copies for each microbial organism in the sample (and therefore the number of cells for said organisms), and the information needed for the equation would be available to the ordinary artisan for all known bacteria in a sample. For example, Mitter teaches using GenBank Accession numbers to determine genomic sequences (and therefore lengths; para. 123 and Table C). As Cutliffe in view of Stämmler establishes the use of bacteria of known amounts being added to the sample as spike-in sequences, these would act similarly to the known sequences of Mitter, and so calibration curves could be made for the spike-in and unknown concentration sequences, where the results could then be used as the DNA quantities described by Mitter, which when combined with fragment length data as also described by Mitter, are analogous to the claimed molecular weights. This then additionally teaches steps (e) and (f) of instant claim 1.
Thus, claims 1 and 4-5 are prima facie obvious over Cutliffe, in view of Stämmler, and in view of Mitter.
Regarding claim 2, this claim further specifies how to calculate cell number, using the information from steps d and e of claim 1. As stated above in the rejection of claim 1, Mitter teaches a formula in paras. 460-461, where said formula differs from the formula of claim 2 only in the stated molecular weight of dsDNA (660 vs 650g per bp). As stated in MPEP 2144.05, similar amounts in the prior art are obvious to those used in the instant invention, particularly in view of the fact that the molecular weight of dsDNA is an approximation. Thus, this difference alone is not enough to render the claimed invention non-obvious over the equation of Mitter. Additionally, Cutliffe, in view of Stämmler, and in view of Mitter, as stated above, also teaches determining values analogous to molecular weights by measuring DNA quantity relation to fragment length, arriving at b of claim 2. The DNA quantity is obtained via calibration curves measurements of known (spike-in) and unknown nucleic acids, based on relative abundance/copy number measurements taken previously in the method, thus arriving at a of claim 2. Thus, Cutliffe, in view of Stämmler, and in view of Mitter also render obvious the method of claim 2.
Regarding claim 7, Cutliffe teaches that the microbiome profile can be taken for a subject at different time points (paras. 11-12). Additionally, Cutliffe teaches comparing the subject microbiome to a reference profile to determine similarities and differences (paras. 81-82). The reference also notes that novel hit databases can be generated based on matches (or lack thereof) between subject and reference sequencing results when performing whole genome shotgun sequencing (para. 135).
Though Cutliffe does not specifically teach excluding known or normal microbial organisms from the microbiome profiles, it would be obvious to do so in the method of Cutliffe, in view of Stämmler, and in view of Mitter in light of the teachings of Cutliffe described above. When comparing microbiome profiles over time or to a reference profile, the ordinary artisan would be looking for differences between the profiles, and could exclude similar organisms between the two profiles (i.e. focusing on the novel hit databases). Thus, this would lead to a closer examination of unknown or non-typical bacteria present in the profile. If a subject has experienced disease progression over time or the sudden onset of disease, the presence of new bacterial species in their microbiome profile may be of great use in determining the cause of such disease, determining diagnoses, or developing treatment plans. There would be a reasonable expectation of success in performing this exclusion as making this change in the method of Cutliffe, in view of Stämmler, and in view of Mitter would simply involve removing data from downstream analyses, and would not involve altering the actual sample collection, spike addition, extraction, or sequencing.
Thus, claim 7 is prima facie obvious over Cutliffe, in view of Stämmler, and in view of Mitter.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Cutliffe et al. (US 2015/0259728 A1), in view of Stämmler et al. (Microbiome, 2016), in view of Mitter et al. (US 2016/0330976 A1), and further in view of Sung et al. (WO 2021/102293 A1).
Cutliffe, in view of Stämmler, and in view of Mitter teach the method of claim 1, as described above. However, none of these references teach the exclusion of unknown or abnormal microbial organisms in the sample.
Sung teaches analysis of stool samples for microbial species and the use of metagenomic data (Abstract). In examining the gut microbiome via stool samples, Sung details that viral or bacterial species that are unclassified, unknown, or of low-prevalence can be excluded from meta-datasets (paras. 88 and 102). Figures 4A also notes that sample exclusion criteria can be based on unclassified taxa or outliers.
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 Sung in the method of Cutliffe, in view of Stämmler, and in view of Mitter to exclude unclassified or low prevalence taxa when establishing the microbiome profile. This would be to avoid the effects of outliers that are less prevalent in the microbiome, which could skew results, and also would better focus the diagnostic assay on bacteria that are known so that targeted treatments could be used. While noting if unknown bacteria are present in a microbiome profile could be useful, it provides no benefit to link them to a particular disease or condition without knowing what the species are, and thus would require assays that are outside the scope of the invention of Cutliffe, in view of Stämmler, and in view of Mitter. Thus, by using the teachings of Sung, the assay of Cutliffe, in view of Stämmler, and in view of Mitter can be streamlined to provide the most accurate and useful clinical information. There would be a reasonable expectation of success in performing this exclusion as making this change in the method of Cutliffe, in view of Stämmler, and in view of Mitter would simply involve removing data from downstream analyses, and would not involve altering the actual sample collection, spike addition, extraction, or sequencing.
Therefore, claim 6 is prima facie obvious over Cutliffe, in view of Stämmler, in view of Mitter, and further in view of Sung.
Conclusion
No claims are currently allowable.
THIS ACTION IS MADE FINAL. 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
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