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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 04/07/2026 has been entered.
Claims Status
Claim 30 is added in the claim set filed on 04/07/2026.
Claims 16-17 and 19-30 are pending and currently under examination
Priority
This application is a national stage application under 35 U.S.C. § 371 of International Patent Application No. PCT/EP2021/059836, filed internationally on April 15, 2021, which claims the priority benefit of French Patent Application No. 2003789, filed April 15, 2020, and European Patent Application No. 20177695.2, filed June 1, 2020
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed on 10/11/2022 for parent Application No. FR2003789, filed on April 15, 2020. The certified copy has been filed on 10/11/2022 for parent Application No. EP20177695, filed on June 1, 2020. An English translation of the foreign application is on record for Application No. FR2003789 and EP20177695. Accordingly, the priority date of instant claims is determined to be April 15, 2020, the filing date of FR2003789.
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.
Claims 16-17 and 19-30 are rejected under 35 U.S.C. 103 as being unpatentable over El Khattabi et al. (“El Khattabi”; (2016). Could digital PCR be an alternative as a non-invasive prenatal test for trisomy 21: a proof-of-concept study. PloS one, 11(5), e0155009.) in view of Hindson et al. (“Hindson”; (2011). High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical chemistry, 83(22), 8604-8610.) and Vogelstein et al. (“Vogelstein”. (1999). Digital pcr. Proceedings of the National Academy of Sciences, 96(16), 9236-9241.)
El Khattabi discloses a method wherein “dPCR-based non-invasive prenatal testing (NIPT) for trisomy 21 has not previously been developed. Here, we designed a Digital PCR as a Non-Invasive Prenatal Test for Trisomy 21 analysis, technical solution to this problem and tested it on plasma DNA samples from pregnant women, in order to establish whether our dPCR technique reliably discriminates between euploid samples and trisomic fetuses (Pg. 2-3, Introduction, last two sentences).
Regarding claim 16, El Khattabi teaches a method wherein “Blood samples were collected ” (Pg. 3, Patient recruitment, Para. 1). El Khattabi teaches a method wherein “By generating uniform water-in-oil droplets, ddPCR partitions the target sequences into approximately 15,000 individual “nanoreactors” in the same well. Following an endpoint PCR using hydrolysis probes, each droplet is read and counted as positive or negative, depending on the presence or absence of target DNA sequence... Absolute quantification is based on the number of positive droplets and Poisson sampling statistics” (Pg. 3, Droplet digital PCR, Para. 1). El Khattabi also teaches a method comprising “We used the QX100 Droplet Digital PCR system …We designed a two-color octoplex PCR experiment that overcomes limitations on the amount of DNA by increasing the number of targets ... We used a set of four FAM TaqMan® hydrolysis assays for the … genes to detect chromosome 21.” (Pg. 3, Droplet digital PCR, Para. 2). Thus, El Khattabi suggests a method comprising: step a) obtaining a sample comprising at least two nucleic acid targets of a genetic event; step b) partitioning the sample from a subject into a plurality of partitions; step c) performing a digital polymerase chain reaction (dPCR) assay on the plurality of partitions under conditions suitable to amplify; and step d) detecting fluorescence in the plurality of partitions, wherein fluorescence in a given partition is indicative of amplification of at least one nucleic acid target of the genetic event in said given partition.
El Khattabi does not explicitly teach the following limitations of step (b): (1) wherein upon spontaneous fragmentation, artificial fragmentation, or a combination thereof of nucleic acids of the sample, the at least two nucleic acid targets are located on separate fragments, and thereby statistically behave independently from each other during the partitioning of the sample into the plurality of partitions, and (2) wherein said partitions comprise an amplification mixture to perform dPCR as described in sub-steps (i-iii).
Regarding the limitations (1-2) of claim 16, Hindson discloses “digital PCR enables the absolute quantitation of nucleic acids in a sample… we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics” (Abstract).
Regarding claim 16, Hindson teaches a method of determination of GRB7 and ERBB2 Copy Number Alterations comprising “Purified DNA… was digested with 0.2 units of NlaIII in 10 μL for 1 h at 37 °C. The restricted DNA was added directly to ddPCR Mastermix” (Pg. 8608, Determination of GRB7 and ERBB2 Copy Number Alterations, Para. 1; Figure 2C). NlaIII is interpreted as a restriction enzyme. “Determination of GRB7 and ERBB2 Copy Number Alterations” reads on at least two nucleic acid targets. Hindson also teaches “Because increases in gene copy number are often the result of tandem gene duplications, we used restriction enzymes to predictably and efficiently separate linked copies of the target gene such that each sequence is encapsulated into its own droplet and counted separately” (Pg. 8606, Col. 2, Para. 2; Figure 2A). Furthermore, Hindson teaches that “The current ddPCR system can achieve read depths of up to 20 000× for two genes from a single well” (Pg. 8606. Col.2, last Para.). Thus, El Khattabi and Hindson suggest a method wherein upon spontaneous fragmentation, artificial fragmentation, or a combination thereof of nucleic acids of the sample, the at least two nucleic acid targets are located on separate fragments, and thereby statistically behave independently from each other during the partitioning of the sample into the plurality of partitions.
Regarding claim 16, Hindson also teaches a method comprising “droplets support PCR amplification … similar to those widely used for real-time PCR applications (i.e., TaqMan)” (Pg. 8605 Col. 1 Para. 3) and “ddPCR Mastermix and TaqMan reagents” (Pg. 8605, Results and Discussion, Para. 1). Hindson teaches a method comprising “duplex TaqMan assay reagents” (Pg. 8606, Col. 2 Para. 3). Hindson teaches a method comprising “The TaqMan PCR reaction mixture was assembled from a 2x ddPCR Mastermix (Bio-Rad), 20x primer, and probes” (Pg. 8608, Droplet Digital PCR Workflow, Para. 1). Hindson teaches a method comprising “The digest was … was assayed per 20 μL ddPCR reaction. … (forward primer) … (reverse primer) ... and (probe) 6FAM-…-MGBNFQ. … All CNV assays were duplexed with an RPP30 reference assay (forward primer)… (reverse primer) … and (probe) VIC-…-MGBNFQ” (Pg. 8608, Determination of Copy Number Variation in HapMap Samples, Para. 1). “FAM” and “VIC read on different fluorescently labeled probes with non-overlapping. Thus, El Khattabi and Hindson suggest a method wherein said partitions comprise an amplification mixture comprising: i. a polymerase; ii. at least two pairs of nucleic acid primers, each pair of nucleic acid primers being capable of hybridizing to a respective one of the at least two nucleic acid targets; and iii. at least two nucleic acid probes, each nucleic acid probe being capable of hybridizing to a respective one of the at least two nucleic acid targets, and being attached or linked to a different detectable fluorescent label.
Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detecting nucleic acid targets as taught by El Khattabi to incorporate the method of fragmenting the nucleic acid sample prior to dPCR with a sequence specific endonuclease and wherein said partitions comprise an amplification mixture comprising: i. a polymerase; ii. at least two pairs of nucleic acid primers, each pair of nucleic acid primers being capable of hybridizing to a respective one of the at least two nucleic acid targets and iii. at least two nucleic acid probes, each nucleic acid probe being capable of hybridizing to a respective one of the at least two nucleic acid targets, and being attached or linked to a different detectable fluorescent label as taught by Hindson and provide fragmentation of the nucleic acid sample and duplex amplification mixture. Doing so would decrease the viscosity of nucleic acids in solution and improve accuracy of nucleic acid region quantitation and copy number variation and could also enhance detection of fetal over maternal DNA, as well as allow for the detection of one or more nucleic acid targets, simultaneously.
Although well known in the art, El Khattabi and Hindson do not explicitly teach the different detectable fluorescent labels having nonoverlapping excitation and/or emission wavelength ranges.
Regarding the limitations (2) of claim 16, Vogelstein discloses “the ultimate utility of Dig-PCR lies in its ability to convert the intrinsically exponential nature of PCR to a linear one. It should thereby prove useful for experiments requiring the investigation of individual alleles, rare variants, mutations, or quantitative analysis of PCR products” (Pg. 9241, Discussion, last para).
Vogelstein teaches a system comprising “primer F1, … primer R1, … Platinum Taq polymerase (Pg. 9236, Materials and Methods, Para. 1) and “molecular beacon (MB)-GREEN, MB-RED… fluorescence was read at excitation emission wavelengths of 485/530 nm for MB-GREEN and 530/590 nm for MB-RED” (Pg. 9236, Materials and Methods, Para. 2). “excitation emission wavelengths of 485/530 nm for MB-GREEN and 530/590 nm for MB-RED” reads on non-overlapping excitation ranges. Thus, El Khattabi, Hindson and Vogelstein suggest a method wherein the different detectable fluorescent labels having non-overlapping excitation wavelength ranges, non-overlapping emission wavelength ranges, or a combination thereof.
El Khattabi, Hindson and Vogelstein are considered to be analogous to the claimed invention because they are in the same field of ddPCR. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detecting at least two nucleic acid targets using dPCR reagents needed for the amplification two nucleic acid targets and detection of fluorescent readout of at least two nucleic acid targets as taught by El Khattabi and Hindson to incorporate the method wherein different detectable fluorescent labels have nonoverlapping excitation wavelength ranges as taught by Vogelstein and provide a method for detecting at least two nucleic acid targets using different detectable fluorescent labels that do not overlap. Furthermore, it would be obvious to the ordinary artisan before the effective filling date to optimize the design of the probes to harbor ideal fluorescent labels to be able to easily distinguish the detectable probes of each individual probe with high sensitivity. Of note, the MPEP states, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). Doing so would allow for the expected increased sensitivity of distinguishing the two separate nucleic acid targets.
The teachings of El Khattabi, Hindson and Vogelstein are documented above in the rejection of claim 16 under 35 U.S.C. 103. Claim 17, 19, 21-22, 25-27 and 29 depend on claim 16. Claim 20 depends on claim 19, which depends on claim 16. Claim 24 depends on Claim 23, which depends on claim 22, which depends on claim 16. Claim 28 depends on claim 27, which depends on claim 16.
Regarding claim 17, El Khattabi teaches a method wherein “The fluorescence signal was measured and analyzed using a QX100 droplet reader and QuantaSoft software” (Pg. 4, ddPCR with circulating cell-free DNA, Para. 1). Thus, El Khattabi, Hindson and Vogelstein suggest a method further comprising measuring the concentration of each nucleic acid target detected.
Regarding claim 19, Vogelstein teaches a method wherein “Dig-PCR is as easily applied to RT-PCR products generated from RNA templates as it is to genomic DNA” (Pg. 9240, Col. 1, Para. 2). A cDNA target is interpreted as product of RT-PCR. Thus, El Khattabi, Hindson and Vogelstein suggest a method wherein each nucleic acid target of the genetic event is an RNA target, and the method comprises a step of reverse-transcribing the RNA target, thereby obtaining at least one reverse-transcribed cDNA target.
Regarding claim 20, Vogelstein teaches a method wherein “Dig-PCR is as easily applied to RT-PCR products generated from RNA templates” and “transcripts from a gene could be determined easily by using fluorescent probes specific for each of the PCR products generated. Similarly, Dig-PCR could be used to quantitate relative levels of gene expression within an RNA population. For this amplification, each well would contain primers that are used to amplify a reference transcript expressed constitutively as well as primers specific for the experimental transcript” (Pg. 9240, Para. 2). Thus, El Khattabi, Hindson and Vogelstein suggest a method wherein the step of reverse- transcribing is carried out after step (b).
Regarding claim 21-24, Hindson teaches a method of determination of GRB7 and ERBB2 Copy Number Alterations comprising “Purified DNA… was digested with 0.2 units of NlaIII in 10 μL for 1 h at 37 °C. The restricted DNA was added directly to ddPCR Mastermix” (Pg. 8608, Determination of GRB7 and ERBB2 Copy Number Alterations, Para. 1; Figure 2C). NlaIII is interpreted as a sequence specific restriction enzyme. “Determination of GRB7 and ERBB2 Copy Number Alterations” reads on at least two nucleic acid targets. Hindson also teaches “Because increases in gene copy number are often the result of tandem gene duplications, we used restriction enzymes to predictably and efficiently separate linked copies of the target gene such that each sequence is encapsulated into its own droplet and counted separately” (Pg. 8606, Col. 2, Para. 2; Figure 2A). Furthermore, Hindson teaches that “The current ddPCR system can achieve read depths of up to 20 000× for two genes from a single well” (Pg. 8606. Col.2, last Para.). Thus, El Khattabi, Hindson and Vogelstein suggest a method wherein the spontaneous fragmentation, the artificial fragmentation, or the combination thereof of the nucleic acids occurs in the sample, before step (b) of the partitioning the sample into a plurality of partitions; comprising, before step (b), artificially fragmentating nucleic acids in the sample in a condition suitable to have the at least two nucleic acid targets located on separate fragments of said nucleic acids; wherein the artificially fragmentating the nucleic acids is carried out by contacting the sample with at least one sequence-specific endonuclease; and wherein the at least one sequence-specific endonuclease is selected from sequence-specific endonucleases capable of cleaving the nucleic acids between the at least two nucleic acid targets.
Regarding claim 25, El Khattabi teaches a method wherein the genetic event is “trisomy
21” (Pg. 5, Linearity regarding the percentage of trisomy 21, Para. 1). Thus, El Khattabi, Hindson and Vogelstein suggest a method wherein the genetic event is at least one of: Down syndrome 21, Edwards syndrome 18, Patau syndrome 13, trisomy 9, tetrasomy 9p, Warkany syndrome 2, cat eye syndrome, trisomy 22, trisomy 16, 1q21.1 deletion syndrome, 1q21.1 duplication syndrome, TAR syndrome, lp36 deletion syndrome, Wolf-Hirschhorn syndrome, cri du chat syndrome, chromosome 5q deletion syndrome, Williams syndrome, Jacobsen syndrome, Miller-Dieker syndrome, Smith-Magenis syndrome, DiGeorge syndrome, 22q11.2 distal deletion syndrome, 22ql3 deletion syndrome, Angelman syndrome, Prader-Willi syndrome, distal 18q-, proximal 18q-, Turner syndrome, Klinefelter syndrome, XXYY syndrome, XXXY syndrome, 49XXXYY syndrome, 49XXXXY syndrome, triple X syndrome, tetrasomy X, 49XXXXX, Jacobs syndrome, 48XYYY, 49XYYYY, 45X/46XY, 46XX/46XY.
Regarding claim 26, El Khattabi teaches a method comprising “pregnant women at high risk of chromosomal abnormalities” and “Blood samples were collected” (Pg. 3, Patient recruitment, Para. 1). Conclusions regarding the presence of the genetic event were provided in Supplemental Table 2 (Supp. Table 2, appended to end of NPL). Thus, El Khattabi, Hindson and Vogelstein suggest a method wherein the sample is a blood sample from a pregnant female subject carrying an embryo or a fetus.
Regarding claim 27, El Khattabi teaches a method wherein “By generating uniform water-in-oil droplets, ddPCR partitions the target sequences into approximately 15,000 individual “nanoreactors” in the same well. (Pg. 3, Droplet digital PCR, Para. 1). El Khattabi teaches a system comprising “Blood samples were collected (in EDTA tubes)” (Pg. 3, Patient recruitment, Para. 1). El Khattabi teaches a system comprising “We designed a two-color octoplex PCR experiment that overcomes limitations on the amount of DNA by increasing the number of targets ... We used a set of four FAM TaqMan hydrolysis assays for the BRWD1, LTN1, NCAM2 and RUNX1 genes … to detect chromosome 21” (Pg. 3. Droplet digital PCR, Para. 2). El Khattabi teaches a system comprising “QX100 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA, USA)” (Pg. 3. Droplet digital PCR, Para. 2). El Khattabi teaches a system comprising “three-step centrifugation protocol was set up for plasma recovery…Circulating DNA was extracted using the QIAampTM Circulating Nucleic Acid Kit (QIAGEN, Valencia, CA, USA)” (Pg. 3, Sample processing and DNA extraction, Para. 1). El Khattabi teaches a system comprising “In a final 20 μL reaction volume, we mixed 10 μL …with 0.5 μL … 2 μL of DNA and water up to 20 μL”(Pg. 4, Validation of the multiplex PCR, Para. 1). “μL” is interpreted as a volume usually measured by pipetting.
Regarding claim 27, Hindson teaches a system comprising “droplets support PCR amplification … similar to those widely used for real-time PCR applications (i.e., TaqMan)” (Pg. 8605 Col. 1 Para. 3) and “ddPCR Mastermix and TaqMan reagents” (Pg. 8605, Results and Discussion, Para. 1). Hindson teaches a system comprising “duplex TaqMan assay reagents” (Pg. 8606, Col. 2 Para. 3). Hindson teaches a system comprising “The TaqMan PCR reaction mixture was assembled from a 2x ddPCR Mastermix (Bio-Rad), 20x primer, and probes” (Pg. 8608, Droplet Digital PCR Workflow, Para. 1). Hindson teaches a system comprising “The digest was … was assayed per 20 μL ddPCR reaction. … (forward primer) … (reverse primer) ... and (probe) 6FAM-…-MGBNFQ. … All CNV assays were duplexed with an RPP30 reference assay (forward primer)… (reverse primer) … and (probe) VIC-…-MGBNFQ” (Pg. 8608, Determination of Copy Number Variation in HapMap Samples, Para. 1). “FAM” and “VIC read on different fluorescently labeled probes with non-overlapping.
Regarding claim 27, Vogelstein teaches a system comprising “primer F1, … primer R1, … Platinum Taq polymerase (Pg. 9236, Materials and Methods, Para. 1) and “molecular beacon (MB)-GREEN, MB-RED… fluorescence was read at excitation emission wavelengths of 485/530 nm for MB-GREEN and 530/590 nm for MB-RED” (Pg. 9236, Materials and Methods, Para. 2). “excitation emission wavelengths of 485/530 nm for MB-GREEN and 530/590 nm for MB-RED” reads on non-overlapping excitation ranges.
Thus, El Khattabi, Hindson and Vogelstein suggest a system comprising “A system for performing the method according to claim 16, the system comprising: - at least one container suitable for storing a sample from a subject; - a subsystem and reactants for performing a digital PCR assay, said reactants including an amplification mixture comprising: i. a polymerase; ii. at least two pairs of nucleic acid primers, each pair of nucleic acid primers being capable of hybridizing to a respective nucleic acid target of the genetic event; and iii. at least two nucleic acid probes, each nucleic acid probe being capable of hybridizing to a respective nucleic acid target of the genetic event, and being attached or linked to a different detectable fluorescent label, the different detectable fluorescent labels having non-overlapping excitation wavelength ranges, non-overlapping emission wavelength ranges, or a combination thereof; and a subsystem for detecting a fluorescence signal.
Regarding claim 28, Hindson teaches a method of determination of GRB7 and ERBB2 Copy Number Alterations comprising “Purified DNA… was digested with 0.2 units of NlaIII in 10 μL for 1 h at 37 °C. The restricted DNA was added directly to ddPCR Mastermix” (Pg. 8608, Determination of GRB7 and ERBB2 Copy Number Alterations, Para. 1; Figure 2C). NlaIII is interpreted as a restriction enzyme that extracting nucleic acids. Hindson also teaches a system comprising “eight-channel disposable droplet generator cartridge” (Pg. 8608, Droplet Digital PCR Workflow, Para. 1). Thus, El Khattabi, Hindson and Vogelstein suggest a method further comprising at least one of: a subsystem and reactants for extracting nucleic acids; a subsystem and reactants for artificially fragmenting nucleic acids; and pipetting means.
Regarding claim 29, El Khattabi teaches a method wherein the genetic event is “trisomy 21” (Pg. 5, Linearity regarding the percentage of trisomy 21, Para. 1). Thus, El Khattabi, Hindson and Vogelstein suggest a method wherein the genetic event is a monosomy, a trisomy or a tetrasomy.
Regarding claim 30, Hindson teaches a method further comprising “For droplets that contain template, specific cleavage of TaqMan probes generates a strong fluorescence signal. On the basis of fluorescence amplitude, a simple threshold assigns each droplet as positive or negative. As the droplet volume is known, the fraction of positive droplets is then used to calculate the absolute concentration of the target sequence.” (Pg. 8605 Col. 2 last line -8606 Col.1 Para. 1). Thus, El Khattabi, Hindson and Vogelstein suggest a method further comprising detecting said genetic event in the sample when, for each nucleic acid target detected, the measured concentration is outside a corresponding predetermined concentration range.
Response to Arguments
Applicant' s arguments filed 04/07/2026 (Pg.7-9) with respect to claims 16-17 and 19-30 have been considered but are not persuasive. To clarify some instances argued in the response filed 04/07/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “Hindson is limited to separating linked duplicates of a single nucleic acid sequence using a restriction enzyme. In contrast, the method of the pending claims requires at least two different nucleic acid targets (e.g., different loci associated with a chromosome abnormality) that are rendered independent through fragmentation. Hindson fails to teach or suggest that a fragmentation results in cleavage between two distinct nucleic acid targets.” (Pg. 9)
Response: In response to applicant's argument stated above, as stated in the Non-final action above, “Hindson teaches a method of determination of GRB7 and ERBB2 Copy Number Alterations comprising “Purified DNA… was digested with 0.2 units of NlaIII in 10 μL for 1 h at 37 °C. The restricted DNA was added directly to ddPCR Mastermix” (Pg. 8608, Determination of GRB7 and ERBB2 Copy Number Alterations, Para. 1; Figure 2C). NlaIII is interpreted as a restriction enzyme. “Determination of GRB7 and ERBB2 Copy Number Alterations” reads on at least two nucleic acid targets. Hindson also teaches “Because increases in gene copy number are often the result of tandem gene duplications, we used restriction enzymes to predictably and efficiently separate linked copies of the target gene such that each sequence is encapsulated into its own droplet and counted separately” (Pg. 8606, Col. 2, Para. 2; Figure 2A).” Furthermore, Hindson teaches that “The current ddPCR system can achieve read depths of up to 20 000× for two genes from a single well” (Pg. 8606. Col.2, last Para.). Thus, Hindson does suggest that a fragmentation results in cleavage between two distinct nucleic acid targets.
Conclusion
No claims are in condition for allowance.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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