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 .
Election/Restrictions
Applicant’s election without traverse of the following in the reply filed on 03/05/2026 is acknowledged:
Group 1, claims 1-19; and
The specific set of amplification set comprising “zero of (iii), (iv), and (v)”.
Claims 2, 6, and 15-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention or species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 03/05/2026.
The request for rejoinder of the remaining subject matter should the elected species be found patentable is noted.
Status of Claims
Claims 1-20 are pending in the instant application, claims 2, 6, and 15-20 have been withdrawn, and claims 1, 3-5, and 7-14 are the subject of this non-final office action.
Specification
The use of the terms including “CY” (e.g., CY3), “ALEXA”, “OREGON GREEN”, “TEXAS RED”, “ATTO”, each which is a trade name or a mark used in commerce, has been noted in this application. Such terms should be accompanied by the generic terminology; furthermore, such terms should be capitalized (i.e., each letter) wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
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.
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.
Claim 5 is 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 5, the claim recites “the Y chromosome”. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1, 3-5, and 8-14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gromminger (US 2017/0314073 A1; published 11/02/2017) in view of Jensen (US 2022/0403468 A1; filed 06/08/2021).
Regarding claim 1, Gromminger teaches a method comprising a digital amplification reaction method (para [0047-76], [0087]; Example 1-2; Fig. 2;):
(a) partitioning into partitions (para [0060], [0069], [0076]) an amplification reaction mixture comprising cfDNA from a cfDNA sample, amplification reagents, and a plurality of amplification sets comprising primer and probe sets (para [0057], [0065-69], [0070]), wherein each amplification set comprises primers and probes for multiplex amplification and each amplification set generates amplification products, when target is present (para [0065], [0067], [0070]), comprising a distinct label distinguishable from the label for each of the other amplification sets (para [0060], [0063], [0067], [0087]; Fig. 2; see also para [0007] and [0194]); and wherein the plurality comprises:
(i) an amplification set that targets sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA (para [0079])
(b) incubating the cfDNA with a methylation-sensitive restriction enzyme (MSRE) cocktail comprising at least one methylation-sensitive restriction enzyme that cleaves unmethylated DNA (para [0056-57]; see also [0070] and [0180]);
(c) amplifying target nucleic acid sequences in the partitions, if present, to obtain amplification products (para [0057] and [0065-69]; see also para [0183] and Table 2);
(d) detecting in the partitions a signal from each distinct label from the amplification products (para [0061-63]; see also para [0067-69] and [0183]); and
(e) quantifying the signal for each distinct label (para [0061-63]; see also para [0067-69] and [0123]),
wherein the method may be used to estimate the fetal fraction (para [0106], [0187], [0194-196]) and wherein the cfDNA sample may be obtained from a blood sample from a pregnant human subject (para [0078]; claim 14; see also para [0129]).
Gromminger teaches various degrees at which sites may be considered to be differentially methylated including about 10%, 20%, 80%, and 90% (para [0054]).
It is noted that Gromminger teaches multiple sites per DMR as sited above. Gromminger also teaches multiple DMRs (e.g., para [0072]) [i.e., for the set that targets hypermethylated fetal DNA] and that utilizing one detectable moiety for all probes has improved detection (Example 2).
Gromminger teaches an embodiment with two species of DNA that originates from cells of a fetus and/or the placenta and that are distinguished from their counterpart of maternal origin in the sample due to differential DNA methylation (para [0129]).
Regarding claims 3 and 4, Gromminger teaches at least three sites methylation sites for an amplification set (para [0070]; Fig. 1) and designing a primer pair and probe for each (para [0070; Fig. 2), wherein each probe of the set is labeled with the same label as discussed and cited above.
Regarding claim 5, Gromminger teaches locating at least one of the DMRs of the fetal hypermethylation set in the Y chromosome (para [0080]), such that at least one corresponding “other region”(OR) of the invention would be within the same portion of the genome (i.e., on the Y chromosome; see para [0064] and Figs. 1-2), wherein Gromminger describes such other regions as methylation insensitive (para [0182]).
Gromminger teaches detecting at more than one OR (para [0064]).
Regarding claims 8 and 9, Gromminger teaches that the digital amplification reaction method may be digital droplet PCR (para [0060] and [0069]).
Gromminger teaches ddPCR is high-throughput and permits absolute quantification of copy number (para [0011]).
Regarding claim 10, Gromminger teaches simplifying method to provide for more efficient and accurate detection especially quantitative detection embodiments, including by using a single vessel such as a droplet in oil/water emulsion (para [0060]).
Gromminger teaches that the artisan will readily be able to design PCR primers for the method using known design algorithms/programs (para [0065]).
Gromminger teaches that the PCR primers may overlap methylation sites (para [0070]) and that the use of such MRSEs will be well known to the POSITA (para [0101]).
Regarding claim 11, Gromminger teaches using primers that span CpG methylation sensitive restriction enzyme sites and primers that do not span any restriction enzyme sites (para [0181]).
Gromminger teaches incubation and optionally deactivating enzymes at a higher temperature following incubation (para [0057]).
Regarding claim 12, Gromminger teaches using four or more methylation-sensitive enzymes (para [0070], [0100]), including the enzymes HhaI, HpaII, AciI, HpyCH4IV, and BsaH1 (para [0099]).
Regarding claim 13, Gromminger teaches the cfDNA may be a blood fraction such as plasma or serum (para [0097]).
Regarding claim 14, Gromminger teaches if three DMRs are used, then three other regions are used (para [0072]).
Gromminger teaches that diagnostic accuracy was increased when methylation of multiple genes was analyzed in combination (para [0007]).
Gromminger fails to explicitly teach:
an embodiment with an amplification set comprising both (i) and an amplification set that targets sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA (claim 1);
an order of steps for the MRSE addition in the partitioning (claims 10 and 11); and
determining a normalized copy concentration for each of the targets based on the number of targets in an amplification set (14).
Jensen teaches a collection of primer pairs for identifying the presence of absence of differentially methylated loci, including hypermethylated and hypomethylated loci (para [0204]), wherein the collection of primer pairs may be for identifying the presence or absence of a hypomethylated locus are prepared by a process comprising selecting one or more genomic loci wherein each locus comprises three or more features selected from: (iv) at least 5 CpG methylation sites, (v) a plurality of restriction endonuclease recognition sites … recognized by one or more methylation sensitive restriction endonucleases, (vii) a locus comprising a methylation status of 40% or less in fetal nucleic acid, (viii) a locus comprising a methylation status of 60% or more in maternal nucleic acid, and (ix) a locus comprising a difference in methylation status of 5% or more between fetal nucleic acid and maternal nucleic acid (para [0206]).
Jensen teaches an additional collection of amplification primer pairs for identifying the presence of absence of hypermethylated locus using the same characteristics except using (v) a locus comprising a methylation status of 60% or more in a minority nucleic acid species, (vi) a locus comprising a methylation status of 40% or less in a majority nucleic acid species, and (vii) a locus comprising a difference in methylation status of 5% or more between a minority nucleic acid species and a majority nucleic acid species (para [0206]).
Jensen alternatively teaches using “low methylation” as <80% methylated (para [0103]) and recites that a hypomethylated site may instead be less than 20% and hypermethylated may be greater than 80% and using additional thresholds for each (para [0114]).
Jensen teaches that leveraging the global hypomethylation of the placenta has utility (para [0432]), and that the fetal portion of ccf DNA in pregnant plasma is derived from the placenta (para [0425]). Jensen teaches identifying large “placenta hypomethylated domains” characterized by high, largely invariant levels of DNA methylation in samples of ccf DNA from non-pregnant samples [analogous to maternal sources] and placenta hypomethylation (para [0427]).
Jensen teaches a method of amplifying target polynucleotides comprising such selection of said primer pairs and contacting with said primer pairs to determine a ratio [e.g., fetal fraction] (claim 79; para [0133]; see claim 89), where the amplification may be digital PCR (para [0242]; see also claim 90).
Jensen teaches normalizing a determined value/data (claim 92; para [0220], [0277]) and that normalizing may allow the artisan to minimize data differences, diminish the importance of outlying data, or to bring data to a common scale (para [0279]; instant claim 14).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized DMR amplification primer sets that further include hypermethylated maternal/hypomethylated fetal targets in the method of amplifying DMRs of Gromminger (instant claim 1), motivated by the teachings of Jensen of the utility and invariant nature of these placental [i.e., fetal] hypomethylation regions for detection methods, where such are further taught as primer sets for the same purpose and further would be obvious to combine as equivalents for the same purpose of detecting DNA admixtures of fetal and maternal DNA in samples such as cfDNA, as discussed in MPEP 2144.06.
In so far as the instant specification defines a particular rate of hypomethylation and hypermethylation that may be interpreted to conflict with certain embodiments of Gromminger and Jensen, the rate at which a differentially methylated site may be considered hyper-/hypomethylated would be considered to be a results effective variable such that adjusting the threshold(s) would be obvious to the POSITA before the EFD of the claimed invention, at least because both Gromminger and Jensen teach a wide range of potential percentages and at least Jensen teaches use multiple thresholds in the examples. Obviousness of routine optimization is further discussed in MPEP 2144.05.
It further would have been obvious to the POSITA before the EFD of the claimed invention, if motivated by the teachings of Gromminger to simplify the method to improve at least the efficiency, to include the MRSE(s) in with the amplification reagents such that the incubation would occur before the amplifying and after partitioning (instant claim 10). As taught by Gromminger, it would have been predictable for the artisan to modify any primers to avoid fully spanning restriction sites sequences chosen well-known and predictable MRSEs. It is further noted that such changes in the sequence of adding ingredients are obvious absent unexpected results, as discussed in MPEP 2144.04(IV)(C) and that such modifications are also obvious at least at least as omission of an undesired element, as further discussed in MPEP 2144.04(II)(A).
Alternatively, it would have been obvious to the POSITA before the EFD of the claimed invention to have added the MRSEs prior to the partitioning and to have incubated them such that they could be deactivated prior to the addition of any primers that overlap the restriction enzyme sites (instant claim 11) , as taught by Gromminger. As discussed above, changes to the order of adding ingredients are obvious absent unexpected results.
At least, given the teachings of Gromminger to use the same number of across its analogous “sets,” it would have been obvious to the POSITA before the EFD of the claimed invention to have normalized the copy concentration for each of the targets based on the number of targets in the amplification set sharing the same probe label in the determination in the combined method (instant claim 14), motivated by the teachings of Jensen to normalize determined values/data to minimize data differences and to bring data to a common scale, as the artisan would understand that such normalization would fulfill the same purpose as maintaining the same number across the “sets” of Gromminger but allow the artisan more flexibility.
In so far as Gromminger may be interpreted not to be broadly directed to a digital amplification, such be an obvious species of the genus of amplification and further obvious to the POSITA before the EFD of the claimed invention to have chosen for the amplification of the combined method, motivated by the teachings of Gromminger that ddPCR is high-throughput and capable of absolute quantification.
There would have been a strong expectation of success as both Gromminger and Jensen are directed to methods of detecting and determining amounts of DNA from admixtures in a non-invasive manner, particularly from cell-free DNA and using fetal/maternal methylation levels of DNA species, wherein such a combination represents the application of known techniques to known methods in a predictable manner. Likewise, such modifications of the methods Gromminger in the combined method where Gromminger teaches that such are within the skill set of the ordinary artisan would be predictable.
Claim(s) 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gromminger (US 2017/0314073 A1; published 11/02/2017) in view of Jensen (US 2022/0403468 A1; filed 06/08/2021) as applied to claims 1, 3-5, and 8-14 above, and further in view of Arroyo (Arroyo K, et al. Development of a Droplet Digital™ PCR DNA methylation detection and quantification assay of prenatal tobacco exposure. Biotechniques. 2022 Apr;72(4):121-133. Epub 2022 Mar 8.).
Regarding claim 7, in the combined method, Gromminger teaches that the DMRs may be associated with chromosomal aneuploidy (entire document, e.g., para [0081]).
Gromminger teaches one or more control procedures, wherein the third class of DNA region acts as a control for the modification step, i.e., for the restriction enzyme digestion wherein the control region is amplified and detected by a third set of primer/probe(s) with a third detectable label used for such class of region (para [0077]).
Gromminger teaches using a standard sample of DNA of known amount/concentration that may be readily available from commercial sources (para [0103]).
Gromminger teaches that cleavage of a [methylation specific] restriction enzyme’s recognition sequence may be blocked when a particular based in the enzyme’s restriction site is methylated (para [0056]).
Gromminger and Jensen fail to explicitly teach using a synthetic control target that is completely methylated and/or completely unmethylated.
Arroyo teaches a method comprising ddPCR for DNA methylation detection (Abstract).
Arroyo teaches designing and purchasing synthetic gBlock gene fragments from a commercial source in either fully methylated or fully unmethylated (pg. 123, Control gBlocks gene fragments). Arroyo teaches mixing gBlocks and detecting in a multiplex reaction (pg. 127, para 2; Fig. 3). Arroyo teaches that the concentration was known (Table 2).
Therefore, it would have been obvious to the POSITA before the EFD of the claimed invention to have utilized synthetic gBlock gene fragments, as taught by Arroyo, as a third class of DNA region in the combined method. Such synthetic DNA represents an obvious and predictable species of the genus of a standard sample of DNA of known concentration readily available from commercial sources, as taught by Gromminger, that the artisan would have been motivated to use as positive/negative controls to assess the methylation-sensitive restriction enzyme(s) of the method, as taught from Gromminger.
There would have been a strong expectation of success as such represents the substitution of one known element for another to obtain predictable results.
While not required for the combination, it also is noted that Gromminger teaches that it will be apparent to the artisan that they may may perform methylation-specific PCR using a combination of bisulfite-modified sites and restriction enzymes the recognition site of which is created upon such bisulphite-modification (para [0101]).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Emma R Hoppe whose telephone number is (703)756-5550. The examiner can normally be reached Mon - Fri 11:00 am - 7:00 pm.
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/EMMA R HOPPE/Examiner, Art Unit 1683
/NANCY J LEITH/Primary Examiner, Art Unit 1636
