DETAILED ACTION
Response to Amendment
The amendment filed 08/19/2026 is acknowledged. As a result of the amendments, claims 1-10 and 14-22 are pending; claims 11-13 have been canceled. The limitations of the cancelled claims have been incorporated into claim 1.
Regarding the Office Action mailed 03/24/2026, the rejection of claims 1-10 and 14-22 under 35 USC § 103 withdrawn in lieu of a modified rejection below, further relying on Silver. Applicant’s remarks will be addressed following the rejections. The rejection of claims 11-13 are withdrawn as the claims have been canceled.
Information Disclosure Statement
The Ludlow reference listed on the IDS was not provided. Specifically, the reference provided is a pre-print version, not the actual document cited on the IDS. Therefore, this reference has been crossed out on the IDS as it fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed.
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 1-10 and 14-22 are rejected under 35 U.S.C. 103 as being unpatentable over Loughran [US 20190323095 A1; previously cited] in view of Brooks [Brooks et al., Basics of Enzymatic Assays for HTS. In: Assay Guidance Manual, 2012; previously cited] and Silver [Silver J, et al. Nucleic acids research. 1993 Jul 25;21(15):3593].
Loughran describes a technique for performing absolute quantification of reverse transcription enzymes in a sample which combines product-enhanced reverse transcriptase (PERT) assays and droplet digital PCR (ddPCR) [Loughran, abstract]. PERT assays are used to detect reverse transcriptase (RT) activity of a sample by converting a known amount of RNA template to cDNA and amplifying the cDNA using product specific primers. Since no exogenous RT is added, any accumulated product will be the result of RTs which are endogenous to the sample [Loughran, 0124]. In ddPCR, a PCR solution is partitioned into small reaction volumes in which PCR is performed allowing for enhanced detection of lower abundance molecules. Each of these partitions is read in a binary manner as to whether or not target DNA is present allowing for absolute counting of the presence of individual DNA species [Loughran, 0126, 0127, 0170]. This combined methodology, termed ddPCR, can be used for a number of purposes including quantifying viral vectors or viral production [Loughran, 0158]; providing quality control in the production of vaccines, recombinant proteins, antibodies, or other medical products which may be contaminated by retroviruses [Loughran, 0159]; detecting the viral load from viruses such as HIV [Loughran, 0160]; and in drug discovery to monitor the production of retroviruses in model systems [Loughran, 0161].
To perform ddPERT, a sample is first subjected to lysing conditions and diluted 10-fold with water. This sample mix is then combined with master mix, MS2 phage RNA (i.e., RNA template), RNAse inhibitor, and a primer/probe mixture to generate a reaction mix [Loughran, 0004, 0027, 0175]. While Loughran used a specific master mix, they state that any buffer could be used provided that absolute quantification was not desired. This would be particularly useful if studying the effects of reaction components on RT activity [Loughran, 0178].
Occurring either prior to or after droplet generation, the reaction mix is incubated at 0-42oC for 0-90 minutes (i.e., a set synthesis period) during which time any endogenous RT will synthesize cDNA [Loughran, 0179]. Once the set synthesis period is complete, the sample is partitioned. However, droplet formation can take anywhere from 24-30 minutes, and RT activity would continue in the unpartitioned sample resulting in differential product generation. To combat this, the RT can be inactivated prior to droplet formation by incubating the sample at 65oC for 20 minutes [Loughran, 0180]. Once partitioned, the sample is then subjected to PCR cycling [Loughran, 0184-0190]. After completion of PCR, each droplet is individually flowed past a detector where fluorescence is read and used to determine the number of DNA molecules, or RT enzymes, that were present in the original sample [Loughran, 0191].
Loughran performed this assay using two different RTs, M-MuLV and AMV, and determined that M-MuLV generates cDNA at a much faster rate over time than AMV. Furthermore, Loughran states that combining the commercially defined enzyme unit of a RT (i.e., the amount of enzyme required to incorporate 1nmol of dTTP into an acid-insoluble form in specific reaction conditions) with the results of ddPERT allows for the determination of how much activity is present in single RT molecules [Loughran, 0198-0200].
Loughran does not teach maintaining the RNA template at a non-limiting concentration during cDNA synthesis wherein cDNA accumulates at a substantially constant rate.
Silver states that RT-PCR is conventionally used for detection of minute amounts of RNA template by providing excess exogenous RT to a sample to convert endogenous RNA into amplifiable DNA. They reasoned that this principle could be flipped in order to assess minute amounts of RT activity. In pursuit of this, they developed an RT-PCR assay for sensitively assessing reverse transcription enzyme activity by providing to a sample an excess of exogenous RNA template but no additional RT [Silver, p1]. To test the sensitivity of the assay, they used various amounts of purified RT in a reaction mix containing 21ng of BMV RNA and were able to detect enzymatic activity in mixtures containing as few as 3 RT molecules [Silver, Figure 1].
Brooks discusses enzyme biochemistry and the kinetics of enzyme action as they relate to enzymatic assays, specifically detailing the importance of performing such assays under initial velocity conditions [Brooks, abstract, p3]. Initial velocity is defined as the initial linear portion of the enzyme reaction when less than 10% of the substrate has been depleted. During this period (i.e., set synthesis period), the velocity of the reaction does not change over time and the substrate is present in excess at a concentration that does not significantly change [Brooks, p3]. In order to maintain initial velocity conditions, a large excess of substrate over enzyme is used wherein the ratios can approach one million [Brooks, p7]. Ensuring that enzymatic reactions proceed in initial velocity conditions minimizes the influence of product inhibition, substrate limitation, reverse reactions, and enzyme inactivation on said reaction [Brooks, p4].
Loughran relies on the amount of cDNA produced during a set synthesis period to determine the amount and activity of RT in a sample. Thus, minimizing changes in the availability of the RNA substrate during this period would reduce substrate limitation as a source of variation as described by Brooks. Silver demonstrates that an assay can be configured to provide an excess of exogenous RNA as a template in order to evaluate RT enzyme activity in a sample. Accordingly, one of ordinary skill in the art prior to the effective filing date of the claimed invention would have reasonably expected that applying Silver’s configuration to Loughran’s assay, in order to follow the conventional enzyme-assay principles described by Brooks, would maintain RNA as a non-limiting substrate and provide a substantially constant rate of cDNA production during the selected synthesis period ultimately improving the accuracy and reliability of Loughran’s RT activity assay.
Regarding claim 2, 3, and 5, Loughran teaches that each partition is read in a binary manner to determine if it is positive or negative for a target DNA [Loughran, 0170]. By determining all of the positive partitions and all of the negative partitions, so too is the total number of partitions determined. Similarly, the skilled artisan would be able to take these values and calculate the fraction of positive and/or negative partitions.
Regarding claim 4, Loughran teaches that knowing the commercial definition of an enzyme unit for a particular RT in combination with an absolute count obtained via ddPERT allows one to determine how much activity is present [Loughran, 0200].
Regarding claim 6, Loughran teaches the ddPERT fluorescence results are computer processed and the Poisson distribution is used to determine the number of DNA molecules or RT enzymes that were present. Concentrations are reported as copies per microliter of reaction, or total positives per 20 microliter reaction, wherein a copy refers to a single RT created cDNA molecule [Loughran, 0191].
Regarding claim 7, Loughran states that the first step of their method is to mix a sample with template RNA, nucleotides, at least one primer, and a DNA polymerase to generate a reaction mixture [Loughran, 0004].
Regarding claim 8 and 10, while Loughran uses MS2 phage RNA as their RNA template, they state that any other available RNA template could be substituted, which would include one generated by in vitro transcription [Loughran, 0176].
Regarding claim 9, Brooks teaches that typical ratios of substrate to enzyme are greater than 100 but can approach 1 million [Brooks, p7] and Silver demonstrates this in testing their assay in which they were able to detect RT activity in reaction mixtures containing 21ng of RNA and only 3 copies of RT [Silver, Figure 1].
Regarding claim 14, Loughran states that a droplet comprises oligonucleotide primers wherein different primer pairs can have the same or different melting temperatures and can be prepared by a variety of methods [Loughran, 0142].
Regarding claim 15, Loughran performed PCR with the following conditions: 95oC for 10 minutes; 95oC for 30 seconds and 50-60oC for 60 seconds, repeated for 40-50 cycles; 98oC for 10 minutes; and 10oC hold [Loughran, 0185-0189].
Regarding claims 16, 17, and 21, Loughran teaches that each droplet comprises a buffered solution a reagent for performing amplification including probes for fluorescent detection. These hydrolysis probes were designed to allow detection of an amplified product and were labeled on the 5` end with either 6-FAM or HEX with an internal Zen quencher and terminal Iowa Black quencher. Fluorescence occurs when the fluorophore is released from the quencher by the hydrolysis of the probe by a polymerase. As each droplet is flowed past a detector, this fluorescence is detected [Loughran, 0030, 0133, 0191].
Regarding claim 18, Loughran states that in ddPCR, the PCR solution can be partitioned into nanoliter-size samples and encapsulated into oil droplets [Loughran, 0126].
Regarding claim 19, Loughran performed this assay using two different RTs, M-MuLV and AMV, and determined that M-MuLV generates cDNA at a much faster rate over time than AMV. This means that the rate at which cDNA is synthesized depends upon the specific RT activity of the sample (i.e., the RT present) [Loughran, 0199].
Regarding claims 20, Loughran states that their assay can be practiced with any digital PCR system [Loughran, 0122]. Any system performing this assay would necessarily include a plurality of partitions containing cDNA at partial occupancy, a portion of a reaction mix in which the cDNA was generated, and amplification reagents. Furthermore, aspects of claim 20 are construed as product-by-process limitations. As defined by MPEP 2113.I: “[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself.
Regarding claim 22, as previously discussed, Brooks teaches that linear progression curves for enzyme reactions can be achieved if a large excess of substrate over enzyme is used in the reaction, stating that typical ratios of substrate to enzyme are greater than 100 but can approach one million [p4, L1-4; p7, L3-4]. Per [0080] of the instant specification, the linear phase of the reverse transcription reaction is equivalent to the maximum velocity. Therefore, if appropriate concentrations of both substrate and enzyme are used to keep the reaction of Loughran in the linear phase during a pre-determined synthesis period, as described by Brooks, then the reaction will be proceeding at its maximum velocity over the entirety of said synthesis period.
Response to Arguments
Applicant’s arguments filed 08/19/2026 have been fully considered but they are not persuasive.
Applicant’s arguments are summarized as follows:
The Examiner failed to provide reason or articulated motivation for why a person of ordinary skill in the art would have incorporated the stated general enzyme kinetics teaching of Brooks in the end point driven ddPERT assay of Loughran. Loughran was attempting to quantify enzyme presence, not characterize enzyme kinetics or the enzymatic activity of a specific RT enzyme, and would have “no reason to care about enzyme kinetics generally”. Applicant specifically points out that the claimed invention is directed towards characterizing RT activity (e.g. manufacturer quality control) which Loughran does not teach.
Response:
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007).
In this case, one of ordinary skill would have been motivated to minimize factors other than RT activity that could affect cDNA production in order to ensure that correlations could be drawn between the two. Brooks teaches that insufficient template concentration is one such factor and further teaches that an RT assay in which excess template is provided (i.e., non-limiting) would proceed in a linear fashion resulting in cDNA accumulation at a substantially constant rate. These teachings would motivate the skilled artisan to design their RT activity assay to include a non-limiting amount of template in order to achieve those effects. Accordingly, the motivation to modify is not that Loughran sought to characterize RT kinetics. Rather, it is that the skilled artisan would have been motivated to optimize Loughan’s assay based upon the enzyme kinetic teachings of Brooks. In order to further elucidate this point, Silver is provided to show this is a known assay configuration.
In response to applicant's argument that Loughran does not teach characterizing RT activity of a sample for specific purposes such as quality control, a recitation of the intended use of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim. This notwithstanding, Loughran does teach this aspect, specifically stating ddPERT can used for several purposes including providing quality control in the production of vaccines, recombinant proteins, antibodies, or other medical products which may be contaminated by retroviruses [Loughran, 0159].
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Kara N Kovach whose telephone number is (571)272-8134. The examiner can normally be reached Monday - Friday, 9am - 3pm.
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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