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 .
Claims 1-7, 9, 11-13 and 15 are examined on the merits.
Claim Objections-Withdrawn
Claim Objections-New Necessitated by Amendment
Claim 6 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claim Rejections - 35 USC § 112
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.
Withdrawn in view of amendments-Claims 5-7, 11 and 12 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing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.
New Rejection Necessitated by Amendments-Claim 15 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.
Claim 15 recites the limitation "the signal to noise ratio" in line 1. There is insufficient antecedent basis for this limitation in the claim.
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.
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.
Prior Rejection Maintained- Claim(s) 1, 2, 4, 7, 9, 11 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Kool et al. (US PGPub 20170159112) in view of Scholl et al. (US PGPub 20090286222).
The claimed invention is drawn to a method for detecting the presence of a SARS RNA sequence in a sample comprising nucleic acids, the method comprising: contacting the nucleic acid with a reaction mixture comprising: at least one ATP-releasing nucleotide (ARN) having a structure:
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wherein R is where R is any purine or pyrimidine, or an analog thereof that retains an ability to base pair with a complementary nucleotide; and optionally dNTPs, wherein the combination of dNTPs and ARN is sufficient to provide a substrate for all bases present in the sequence of interest; a primer complementary to the target virus RNA; and a reverse transcriptase (RT) that incorporates ARNs; and detecting the presence of ATP released during extension of the target by the reverse transcriptase.
The reverse transcriptase is a MMLV mutant RT. Applicants have not defined what renders the MMLV reverse transcriptase a mutant RT.
The reaction mixture comprises a plurality of primers complementary to the SARS RNA, ATP-depleted dCp4A and dTp4A, dATP and dGTP.
The ATP is detected by a step of containing the reaction mixture with luciferin and an ATP-dependent luciferase enzyme that produces light.
Kool et al. teach ATP releasing nucleotides with the following structure (see figure 1A):
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Kool et al. teach the detection methods of RNA sequences in a sample by subjecting the sample to a reaction mixture comprising a primer, a reverse transcriptase and ATP-releasing nucleotides. [see paragraph 156] The detection is achieved through amplified isothermal reactions. Specific examples of RNA sequences to be detected are viral RNA sequences. [see paragraph 9] Examples of reverse transcriptases include MMLV. [see paragraphs 86] Kool et al. also teach reaction mixture of the methods of the present invention comprises a combination of dNTPs and ARNs that is sufficient to provide a substrate for all bases present in the target polynucleotide. Generally all four deoxynucleotides are present in a reaction mix, where each deoxynucleotide is provided either as a native dNTP, or as an ARN, e.g. deoxyadenosine-5′-tetraphosphate-P4-5′-adenosine (dCppppA), deoxycytidine-5′-tetraphosphate-P4-5′-adenosine (dAppppA), deoxyguanosine-5′-tetraphosphate-P4-5′-adenosine (dGppppA) or deoxythymidine-5′-tetraphosphate-P4-5′-adenosine (dTppppA). [see [paragraph 12] The four deoxynucleotides are provided as two ARNs, and two native dNTPs. Alternatively, the four deoxynucleotides are provided as three ARNs, and one native dNTP, or all four ARNs are present. For any given base, the reaction mixture will usually contain a native dNTP or an ARN, but not both. Surprisingly, a subset of ARNs combined with dNTPs may provide a stronger signal than a reaction with all four ARNs. [see paragraph 13]
Kool et al. also teach that amplification is also a goal of their methods. Since our targets are relatively long RNAs, we hybridize primers at the allelic site being queried. Supplying all four ARNs will then enable a reverse transcriptase enzyme to proceed from the primer end, making DNA all along the RNA target until its end is reached. Every ARN nucleotide addition releases a molar equivalent of ATP, which can subsequently be detected very sensitively. Since the target RNA may extend far downstream of the primer end, dozens, or even hundreds or thousands, of equivalents of ATP will be produced per molecular strand of RNA target. With correctly matched primer, thousands of equivalents of ATP will be produced, whereas with a mismatched primer, much lower signal will result, as the enzyme proceeds poorly past this mismatch. Thus use of two separate primers in two experiments allows for the comparison: experiments in which one primer yields much higher signal than another allows base calling, and the SNP is correctly identified. If they yield almost the same signals, this would indicate a mixture of the two alleles in the sample, such as might occur with heterozygous patients, or with mixed cancerous and normal tissue. A negative control (with no input RNA) shows the background signal. Signals reproducibly above background indicate positive signal showing the presence of the allele being probed. [see paragraph 159]
Kool et al. also teach that released ATP (from the processed ARNs) can be assayed in a qualitative or quantitative analysis, where one equivalent of ATP is released for every deoxynucleotide incorporated from an ARN. Any convenient method for the detection of ATP can be used, as known in the art, including without limitation: luciferase bioluminescence assays, fluorescent dyes, target-responsive aptasensors, and the like. In some such embodiments, the detection reagent(s) is combined with the reaction mixture after the polymerization reaction is substantially complete, e.g. where a desired level of the product of the reaction has accumulated, such as after at least about 15 minutes. [see paragraph 14] With Kool et al. teaching the detection of ATP release after the polymerization rection (which is the extension phase) is substantially complete, such as at least about 15 minutes, meets the claim requirement of claim 7 (the extension reaction proceeds for about 10 minutes).
Paragraph 116: For reporting on DNA polymerase activity with all possible sequences, a full set of four chimeric ATP-linked dinucleotides is needed. We prepared these by modifying a published procedure used previously for end-labeled nucleoside tetraphosphates (Sims et al. Nat. Methods 2011, 8, 575-580.) Tetra- or tri-alkylammonium salts of standard deoxynucleoside monophosphates (dNMPs) were activated with carbonyldiimidazole and then reacted with the alkylammonium salt of 5′-ATP to produce the desired chimeric dimers. These ARNs were purified by HPLC and ion exchange chromatography, yielding products as lyophilized powders in 42-60% yields.
While Kool et al. teach that reaction mixtures comprising reverse transcriptases, primer(s), dNTPS and ATP-releasing nucleotides can be used to transcribe RNA present in a sample into a nucleotide sequence that can be detected and that viral RNA can be detected, they do not teach the primer or primers necessary to detect a virus.
Scholl et al. teach identifying coronavirus, such as SARS, in a sample by: a) providing
a sample comprising coronavirus RNA,
first primers capable of amplifying genomic coronavirus RNA,
second primers capable of amplifying subgenomic coronavirus RNA,
reverse-transcriptase (RT);
v) DNA polymerase; and
vi) dNTPs;
b) extracting said coronavirus RNA from said sample;
c) initiating an RT-PCR reaction in solution with said extracted coronavirus RNA, said first and second primers, said reverse-transcriptase, said polymerase and said dNTPs under conditions such that first and second amplicons are produced, said first amplicon comprising genomic coronavirus RNA, said second amplicon comprising subgenomic coronavirus RNA. [see claims 1 and 2]
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Kool et al. in order to detecting SARS viral RNA using primers specific for the virus with their nucleic acid amplification protocol. One would have been motivated to do so, given the suggestion by Kool et al. that the method be used to detect viral RNA. There would have been a reasonable expectation of success, given the knowledge that SARS RNA can be detecting using a reverse transcriptase, dNTPs and primers specific for the viral RNA, as taught by Scholl et al. Thus the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Response to arguments:
Applicant presents the following arguments in traversal of the rejection:
The claimed method has an unexpected benefit by improved signal to noise ratios by the use of ATP-depleted dCp4A and that there was substantially improved signal:noise ratio of 25:1, which is almost double that of the combination stated by Kool to be optimal. Applicants also state that using an ultra-processive RT, a 6 fold greater luminescence signal relative to Marathaon RT. Lastly, while Scholl et al. does teach detecting coronaviruses, they do not teach or suggest the claimed reaction mixture and the conditions set forth in the present claims.
In response, Kool et al. teach purification of ARNs dTppppA, dCppppA, dAppppA and dGppppA by RPHPLC and DEAE column. [see paragraphs 139-142]. Applicants state in paragraph 42 of the instant specification that “…Various methods known in the art for depletion of ATP may be used for this purpose, including, without limitation, purification with HPLC to separate ATP from the ARN; reaction of the ARN with luciferase and luciferin to selectively consume residual ATP without affecting dCp4A; and the like. In some embodiments the ATP depleted ARN is dCp₄A…” Therefore, the ARNs of Kool et al. are ATP depleted.
With regard to the ultra-processive reverse transcriptase providing a 6-fold luminescence greater signal, it would appear that reverse transcriptase Superscript IV (SSIV) is an a highly processive RT that would be capable of this greater signal. However, the claims do not require the use of reverse transcriptase SSIV and therefore, the claimed methods are not commensurate in scope with the unexpected results presented by applicants. While applicants have given provided two examples of an ultra-processive reverse transcriptase (SSIV, an engineered MMLV mutant RT, and group II intron-encoded RT), these general structural requirement of an ultra-processive RT is not provided. Therefore, the reverse transcriptases taught by Kool et al., such as MMLV, would meet the claim limitations of an ultra-processive RT.
Prior Rejection Maintained- Claim(s) 3 and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Kool et al. as applied to claims 1, 2, 4, 7, 9, 11 and 13 above, and further in view of Hogan et al. (US PGPub 20220195539).
However, Kool et al. do not teach the detection of RNA from a coronavirus, such as SARS-CoV2 and using a primer or primers selected from SEQ ID NO: 1-9.
Hogan et al. teach the detection of SARS-CoV-2 RNA sequences in samples by using a reverse transcriptase, primers and free nucleotides. One example of a primer is SEQ ID NO: 74, which comprises all of SEQ ID NO: 1 of the instant invention. [see paragraphs 10, 64 and 66]
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Kool et al. in order to detect SARS-CoV-2 viral RNA using a primer, such as SEQ ID NO: 1, specific for the virus with their nucleic acid amplification protocol. One would have been motivated to do so, given the suggestion by Kool et al. that the method be used to detect viral RNA. There would have been a reasonable expectation of success, given the knowledge that detecting RNA from a SARS-CoV-2 can also be achieved by using reverse transcriptases, a primer that comprises SEQ ID NO: 1 and free nucleotides, as taught by Hogan et al. Thus the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Response to arguments:
Applicant presents the following arguments in traversal of the rejection:
Hogan et al. does not teach or suggest the specific reaction mixture and conditions presently claimed.
The teachings of Kool et al. in view of Scholl et al. are summarized above, and they render obvious the specific reaction mixture and conditions as claimed. The teachings of Hogan et al. are relied upon to establish that the prior art taught at least one SARS-CoV-2 primer, which includes at least one of the primers presently claimed (SEQ ID NO:1) Therefore, the combined teachings of Kool et al., Scholl et al. and Hogan et al. render obvious the instant invention.
Prior Rejection Maintained) Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Kool et al. in view of Scholl et al. as applied to claims 1, 2, 4, 7, 9, 11 and 13 above, and further in view of NEB Labs (September, 2016).
The claimed invention also requires that the extension reaction temperature is about 55-degrees Celsius.
The teachings of Kool et al. and Scholl et al. are summarized above, however, they do not specifically state that the extension temperature reaction is about 55 degrees Celsius.
NEB Labs provide a summary of reverse transcriptase (RT) activities during DNA generation from RNA sequences. They use an isothermal DNA amplification approach and analyse the activity of the reverse transcriptase activity at temperatures ranging from 30-degrees Celsius up to approximately 55-degrees Celsius (see line graph presented below). Temperatures between about 45 and 55 degrees showed the highest degree of activity of the RT.
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It would have been obvious to one of ordinary skill in the art to modify the methods taught by Kool et al. and Scholl et al. in order to perform reverse transcriptase driven extension at a temperature of about 55 degrees Celsius. One would have been motivated to do so, given the suggestion by Kool et al. that the method be use reverse transcriptase to generate copies of RNA sequences during an isothermal DNA amplification protocol. There would have been a reasonable expectation of success, given the knowledge that reverse transcriptase can achieve better activity at temperatures between about 45 and 55 degrees Celsius, as taught by NEB Labs. Thus the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Response to arguments:
Applicant presents the following arguments in traversal of the rejection:
The claimed temperature of 55-degrees Celsius is not obvious since they observed an unexpected shift when temperature was increased from 50 to 55-degrees Celsius, as presented in figure 3 of the instant application:
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Figure 3 compares the activity of SSIV to MarathonRT.
NEB shows no improvement in activity between 50 and 55 degrees Celsius, in fact there may have been decreased activity.
In response, NEB shows some improvement between 50 and 55 -degrees Celsius, when compared to the horizontal line added by the examiner. It would appear that 52 and 55 degrees Celsius were better than 50 degrees Celsius. Therefore, one of ordinary skill in the art would be motivated to use at least one of those two higher temperatures. In addition, the RTs used in figure 3 are not presently claimed. Therefore, these results are not commensurate in scope with the results observed by applicant at 55 degrees Celsius.
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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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BENJAMIN P BLUMEL whose telephone number is (571)272-4960. The examiner can normally be reached M-F 8-5 EST.
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/BENJAMIN P BLUMEL/Primary Examiner, Art Unit 1671
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