DNDETAILED 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 .
Status of Claims
Currently, claims 1-12 and 14-17 are pending in the instant application. All the amendments and arguments have been thoroughly reviewed but are deemed insufficient to place this application in condition for allowance. The following rejections constitute the complete set being presently applied to the instant Application. Response to Applicant's arguments follow. This action is FINAL.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Any rejection not reiterated is withdrawn in view of the amendments to the claims.
Claim Rejections - 35 USC § 103
Claims 1-4 and 14-17 are rejected under 35 U.S.C. 103 as being unpatentable over Kroh (Kroh et al; Methods, vol 50, pages 298-301, 2010) and Applied Biosystems (Applied Biosystems TaqMan small RNA assay guide, pages 1-30; 2019) in view of Khoury (Khoury et al; June 2020; PerrJ 8:e9004, pages 1-16), Jordan (US Patent 6,017,699), IDT (Integrated DNA technologies - From the internet: “How to store oligonucleotides for greatest stability, pages 1-5; published June 20, 2017), NEB (New England Biolabs - dNTP product specification, pages 1-2, August 2015), and Kramer (Kramer, M; Stem-Loop RT-qPCR for miRNAs; Current Protocols in Molecular Biology, 2011, pages 1-15).
Kroh teaches quantitative RT-PCR of microRNA. Kroh teaches isolation of total RNA from plasma and serum specimens (claims 2-4) (page 300, col 1). With regard to claim 1, Kroh teaches qRT-PCR of miRNAs using the TaqMan-based qRT-PCR kit, which requires an RT reaction comprising H2O, reverse transcription buffer, RNAse inhibitor, dNTPs with dTTP, reverse transcriptase, primers for detection of particular miRNA, and isolated RNA (see page 300). Kroh teaches creating a master mix for the RT reaction. Kroh teaches a second step of quantitative PCR comprising a PCR master mix, primers for qPCR of the miRNA of interest, and cDNA obtained from the RT step. Therefore, with regard to claim 1, Kroh teaches performing assays with a first RT reaction using a master mix and a second PCR reaction also employing a master mix. Further, Applied Biosystems teaches that the water in the RT reaction and the PCR reaction should be nuclease free water (see pages 13 and 17). Applied Biosystems also teaches providing a PCR reaction mix (PCR master mix). Therefore, it would have been prima facie obvious to the ordinary artisan prior to the effective filing date, to include the use of nuclease free water in the method of Kroh as taught by Applied Biosystems and to use RT as well as PCR master mixes (premix).
Regarding claim 1, Kroh and Applied Biosystems do not teach multiplex qRT-PCR reactions with reactions that contain an RT primer for each species to be detected in the same reaction, or PCR reactions that contain a primer pair for each species to be detected in the same reaction. However Khoury teaches that when detecting multiple miRNAs, single plex reactions increase reagent usage and are problematic when using limiting or rare samples (see page 5). Khoury teaches optimization of multiplex qRT-PCR for detection and quantification of multiplex reactions that contain both miRNA and mRNA species, as well as multiplex reactions that contain multiple different miRNA species (see abstract). Khoury teaches RNAmp lends itself to utilizing multiple fluorescently labeled reporter genes to detect more than one RNA species within a single qPCR reaction (page 2, 3rd para; page 3). Khoury teaches that the experimental design for duplex qRT-PCR is more complicated than single plex due to possible overlap in emission spectra of hydrolysis probes. To address this, Khoury teaches using VIC, FAM, and ROX dyes with little to no overlap in their emission spectra (page 4). Khoury compares single plex vs multiplex reactions for detection of miRNA/mRNA species as well as multiple miRNA species from serum (see figure 4). Khoury teaches that the Cq for detection of two miRNAs in a duplex reaction were similar to those from the single plex reaction (figure 4D, page 11). Khoury concludes that the level of different miRNAs can be measured in a single reaction (page 13). Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have modified the miRNA quantification method of Kroh and Applied Biosystems, to include multiplex analysis using master mixes comprising more than one primer or primer pair in situations where more than one miRNA species are present and require quantification in a sample. Khoury provides motivation to conduct multiplex reactions for the benefit of limiting reagent usage as well as in situations where samples are limited. The ordinary artisan would have had a reasonable expectation of success in quantifying levels of more than one miRNA in a single reaction given the demonstration of successful quantification of multiple miRNA in a single reaction taught by Khoury.
Regarding claim 1, Kroh, Applied Biosystems, and Khoury do not teach an RT premix (master mix) and a quantitative PCR premix (master mix) that were previously stored at -20°C or less. However, Jordan teaches PCR reactions where the extracted DNA template is added to a PCR master mix (see col 14, lines 34-40) containing TRIS buffer, dNTPs, primers and enzyme. Jordan specifically teaches that a large batch of the master mix which did not contain template or enzyme (a pre master mix) was prepared and stored in small individual use aliquots stored at -20°C. Jordan teaches that this helps to prevent contamination problems and assay consistency problems. Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, to have prepared premix solutions as taught by Jordan, that contain buffer, dNTPs, and/or primers and to store them at -20°C, in the method of Kroh and Applied Biosystems with a reasonable expectation success. The use of master mixes in conducting RT and PCR steps was routine in the field as exemplified by the art cited (Kroh, Applied Biosystems, Jordan). Additionally, the storage of primers and dNTPs at -20°C is not only exemplified by Jordan, but the art teaches that these are standard storage conditions for these reagents, including being able to freeze and thaw them up to 5 times without affecting stability. For example:
IDT teaches that best practices for storage of oligonucleotides (primers are oligonucleotides) includes storage at -20°C in TE (Tris-EDTA) buffer and nuclease free water, and that they are stable at this temperature for 24 months (page 1). IDT also teaches that oligonucleotide stability was tested after multiple freeze-thaw cycles and that 30 freeze/thaw cycles had no significant impact on oligo stability (see page 3 “Freezing and thawing your oligos will have minimal impact on real time oligo stability).
NEB teaches that dNTPs should be stored at -20°C (page 1).
Kramer teaches preparing master mixes and storing qPCR reagents such as forward primers, reverse primers, and hydrolysis probe in a master mix at -20° for up to 2 years (see page 12). Kramer teaches to refold RT stem loop primers and that the step does not need to be repeated if the primers are never heated above room temperature. Kramer teaches storage of 100uM and working stocks at -20°C (see page 12, bottom para).
Therefore, the artisan of ordinary skill would have had a reasonable expectation of success that these storage conditions, including through multiple freeze/thaw cycles, would be beneficial and stable in the method of Kroh, Applied Biosystems, Khoury, and Jordan, including with the use of stem loop primers.
While the instantly claimed RT premix and PCR premix in claim 1 contain different combinations of reagents, Kroh and Applied Biosystems use premixes for the RT step and the PCR step, where the reagents in the premixes and for the final reactions are added in a different order, illustrating the routine nature of conducting RT and PCR steps with premix (master mix) compositions. Additionally, as set forth in the MPEP 2144.04 IV(C):
C. Changes in Sequence of Adding Ingredients
Ex parte Rubin , 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.).
Therefore, absent secondary considerations, the order and composition of reagent addition is considered prima facie obvious.
Regarding claims 14-17, the prior art of record also provides a reasonable expectation of success that the reagents in the mixes would be stable through multiple freeze/thaw cycles. It is standard practice to thaw solutions containing assay reagents, including nucleic acids on ice (see Applied Biosystems). Additionally, Kramer teaches that different primers can be stored together at -20°C and also teaches that stem loop primers for miRNA RT reactions need not be refolded as long as the primer is never heated above room temperature.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Kroh, Applied Biosystems, Khoury, Jordan, IDT, NEB, and Kramer as applied to claims 1-4 above, and further in view of Gallelli (Galleli et al; Journal of Clinical Medicine, vol 8, 2019).
Kroh, Applied Biosystems, Jordan, Khoury, IDT, NEB, and Kramer do not teach quantification of miR-34a-5p. Gallelli teaches quantification of miR-34a-5p using qRT-PCR and also teaches that the level of expression is associated with the effects of drug treatment for migraine pain in children and adolescents (see abstract, figure 2). Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to use the method of Kroh, Applied Biosystems, Jordan, Khoury, IDT, Kramer, and NEB to quantify miR-34a-5p to monitor the effects of drug treatment for migraine pain in children and adolescents as taught by Gallelli.
Claims 6 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Kroh, Applied Biosystems, Khoury, Jordan, IDT, Kramer, and NEB, as applied to claims 1-4 above, and further in view of Jiao (Jiao et al; Scientific Reports, vol 8, 2018).
Kroh, Applied Biosystems, Jordan, Khoury, IDT, NEB, and Kramer do not teach the use of spiked in C elegans miRNA controls. However, Jiao teaches the use of cel-miR-40-3p miRNA spike in controls for miRNA analysis and normalization.
Therefore it would have been prima facie obvious to the ordinary artisan prior to the effective filing date to include spike in control cel-miR-40-3p in a single multiplex reaction in the method of Kroh, Applied Biosystems, Jordan, Khoury, IDT, NEB, and Kramer in view of the routine nature of miRNA expression analysis as exemplified by the art cited as well as to accurately monitor gene expression of miRNAs of interest. The ordinary artisan would have had a reasonable expectation of success in arriving at the claimed methods in view of the cited prior art of Jiao and Khoury.
Claims 8-12 are rejected under 35 U.S.C. 103 as being unpatentable over Kroh, Applied Biosystems, Khoury, Jordan, IDT, NEB, and Kramer as applied to claims 1-4 above, and further in view of Gallelli and Jiao.
The teachings of Kroh, Applied Biosystems, Khoury, Jordan, IDT, NEB, and Kramer is set forth above. Kroh, Applied Biosystems, Khoury, Jordan, IDT, NEB, and Kramer do not teach quantification of miR-34a-5p or the use of spiked in C elegans cel-miR-40-3p miRNA controls. However, Gallelli teaches quantification of miR-34a-5p using qRT-PCR and also teaches that the level of expression is associated with the effects of drug treatment for migraine pain in children and adolescents (see abstract, figure 2). Further, Jiao teaches the use of cel-miR-40-3p miRNA spike in controls for miRNA analysis and normalization. Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to use the method of Kroh, Applied Biosystems, Khoury, Jordan, IDT, NEB, and, Kramer to quantify miR-34a-5p to monitor the effects of drug treatment for migraine pain in children and adolescents as taught by Gallelli and to use cel-miR-40-3p spike in control miRNA, as taught by Jiao for the purpose of accurately monitoring gene expression of miRNAs of interest.
Claims 10-12 recite particular concentrations for the reagents in the RT and PCR premixes whose exact ratios are not recited in the prior art. However, absent secondary considerations, these concentrations/ratios are considered prima facie obvious over the teachings of the prior art, which provide detailed instructions on how to make premixes, master mixes, including the use of 10X solutions diluted down in the final mix volume (see Kroh, Applied Biosystems, Jordan, Kramer). As set forth in the MPEP (2144.05 IIA) regarding Optimization with prior art conditions or through routine experimentation:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages."); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.). For more recent cases applying this principle, see Merck & Co. Inc. v. Biocraft Lab. Inc., 874 F.2d 804, 809, 10 USPQ2d 1843, 1848 (Fed. Cir. 1989), cert. denied, 493 U.S. 975 (1989)(Claimed ratios were obvious as being reached by routine procedures and producing predictable results); In re Kulling, 897 F.2d 1147, 1149, 14 USPQ2d 1056, 1058 (Fed. Cir. 1990)(Claimed amount of wash solution was found to be unpatentable as a matter of routine optimization in the pertinent art, further supported by the prior art disclosure of the need to avoid undue amounts of wash solution); and In re Geisler, 116 F.3d 1465, 1470, 43 USPQ2d 1362, 1366 (Fed. Cir. 1997)(Claims were unpatentable because appellants failed to submit evidence of criticality to demonstrate that that the wear resistance of the protective layer in the claimed thickness range of 50-100 Angstroms was "unexpectedly good"); Smith v. Nichols, 88 U.S. 112, 118-19 (1874) (a change in form, proportions, or degree "will not sustain a patent"); In re Williams, 36 F.2d 436, 438, 4 USPQ 237 (CCPA 1929) ("It is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means, is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions."). See also KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416, 82 USPQ2d 1385, 1395 (2007) (identifying "the need for caution in granting a patent based on the combination of elements found in the prior art.").
Response to Arguments
The response traverses the rejection and asserts that the experimental results of the present application show that only the RT premix of the invention, RT-Premix 3 produced reverse transcription of different concentrations of miR-34a at a level most similar to a classical preparation, regardless of storage time. The response asserts that the results show that not all premixes can provide the same level of efficiency in RT-PCR as that achieved with classical preparation and that such would not have been expected (are unpredictable). This argument has been thoroughly reviewed but was not found persuasive because the response does not address the scope of the claims in relation to the scope of the alleged unexpected results. If not all premixes can provide the same level of efficiency, then it appears that broad scope of the claims is not commensurate in scope with the unexpectedly efficient RT-Premix discussed in the response. The previous office actions have addressed the possibility of secondary considerations, and also made clear that nexus is required between the scope of the claims and that of any unexpected results. Any arguments against the references individually remain unpersuasive because the rejection relies on the combined teachings of the references. 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). For these reasons and the reasons already made of record in previous office actions, the rejections are maintained.
Conclusion
No claims are allowed.
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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/JEHANNE S SITTON/Primary Examiner, Art Unit 1682