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 .
DETAILED ACTION
Status of the Claims
Claims 1-9, 11-13 and 15-25 are pending and the subject of this FINAL Office Action.
Allowable Subject Matter
The only allowable subject matter in the specification that the Examiner could identify was the LNA primer sets in Example 6.
Priority
Claims 18-19, 22 and 24 receive a priority date of 11/10/2021 because the priority document (PCT/EP2021/081230) filed on this date is the first priority document to disclose PAR or 5-Br-PAPS.
New Grounds of 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.
Claim(s) 1-8, 11-13, 15, 20 and 25 is/are rejected under 35 U.S.C. 103 as being unpatentable over LIU (US 2017/0362669).
As to claims 1, 11, 13, 15, 20 and 25, LIU teaches methods and kits for performing LAMP using B3 primers that have LNA at their last third of 5’-side (AceIN-B3a_L and AceIN-B3bL used in LAMP sets 31, 43 and 44; Tables 1-3). The LAMP reaction include nucleotides (Examples; para. 0007) and DNA polymerase (e.g. para. 0007).
As to claims 2-3, this only further describes the common LAMP primers F3, B3, BIP and FIP, which are taught in LIU (Table 1).
As to claim 4, LIU teaches RNA or DNA (HIV nucleic acids; Examples).
As to claims 5-6, LIU teaches 15-30 bases with 1-5 LNA (Table 1).
Although LIU does not explicitly teach a LAMP F3 primer with first third from 5’-3’ having LNA, yet LIU demonstrates that optimizing in this way was obvious. Specifically, LIU teaches that placing LNAs at different positions within a LAMP primer yields various amplification efficiencies based on the target. LIU even placed LNAs at different parts of F3 primer ACeIN-F3_cL (CCTATTTGGAAAGG+ACCAGC). LIU explains that the placement of the LNA is dependent on the target, and must be optimized using routine optimization techniques:
Examples 3—Testing Different Primer Designs
ACeIN-1 (“Ace” for “all clade”, and “IN” for “integrase”), targeted the HIV IN coding region and contained multiple bases at selected sites to broaden detection (FIG. 1). ACeIN-2 and -3 have primers with slightly different landing sites. Tests showed that the mixture of primers allowed amplification with a shorter threshold time than did either alone (FIG. 1).
A new primer set was designed to target the CA coding region (FIG. 1, ACeCA) but found that the set only amplified clade B, and not efficiently. ACeIN3-6 were altered by inserting a polyT sequence between the two different sections of FIP and BIP in various combinations, a modification introduced with the goal of improving primer folding, but these designs performed quite poorly (FIG. 1).
Because the FIP primer appeared to bind the region with most variability among clades, variations that bound to several nearby regions were tried. These were tried with and without the polyT containing BIP and FIP primers in various combinations (FIG. 1, ACeIn7-22). All of the variations of FIP were mixed together (ACeIN-23; Table 1, row 29). The ACeIN-23 primer set was tried as a mixture with the B-PR set to try to capture clade F, yielding a relatively effective primer set (FIG. 1, ACeIN23+B-PR).
In an effort to increase affinity, an additional G/C pair was added F3 and tested with various other IN primers (FIG. 1, ACeIN24-31). Testing showed improvement, with ACeIN-26 showing particularly robust function.
In a second effort to increase primer affinities, locked nucleic acids were added to selected primers binding some of the most conserved bases (FIG. 1, ACeIN 30, 31, 32, 33, and 34). Some improvement was shown over the non-LNA containing bases. However, the ACeIN-26 primer set was as effective as or better than any LNA containing primer sets.
In further tests, the ACeIN-26, 28 and 30 primers were tested combined with the B-PR primer (a slightly modified version of the row 3 primer) but no improvement was seen and efficiency may even have fallen for some subtypes. A primer set was also designed that matched exactly to the problematic subtype F, and this set was mixed with the ACeIN-26 primers. However, no improvement was seen (FIG. 1, mixtures with F-IN set). Mixing the ACeIN-26 primers with both the B-PR (redesigned) and F-specific primers did yield effective primer sets (FIG. 1, ACeIN26+F-IN and ACeIN26+F-IN+ACePR). However, this was not greatly improved over the ACeIN-26 primer set, so the simpler ACeIN-26 primer set was used in further studies.
Thus, LIU clearly demonstrates that optimizing LNA placement and numbers in LAMP primers such as F3 primer is an obvious optimization parameter.
As to claim 7, “regulator for melting temperature” is undefined and encompasses anything that affects a temperature (e.g. salt).
As to claim 8, LIU teaches fluorescent detection (Examples).
Applicants argue the Reply 08/10/2026 that LNAs at first third of F3 primer achieve unexpected results. However, the results presented are for a single target (SARS-CoV-2), using s single reaction mixture and single reaction conditions (Examples). In fact, as Applicants seem to acknowledge, the context is critical to the results. Applicants argue at pages 13-14 that “due to the complex nature of primer interactions during LAMP target amplification, changes to primer base composition would not be expected to lead to beneficial effects during LAMP amplification, and may facilitate non-specific interactions and amplification and impede strand displacement activity of the polymerase.” But LIU demonstrates that a skilled artisan would understand this. This is why LIU tested (AKA optimized) various LNA placements in various primers. Primer testing is the epitome of obviousness; this includes LNA placement.
In sum, Applicants must demonstrate that any first-third F3 LNA placement for any target and any reaction conditions yield unexpected results versus F3 the primers of LIU.
Claim(s) 7-9, 11-13 and 16-25 is/are rejected under 35 U.S.C. 103 as being unpatentable over LIU (US 2017/0362669), in view of TANNER (US20210285065) and HIGURASHI (US 20090017545).
The prior art as a whole demonstrates that it would have been obvious to a skilled artisan at the time of filing to use familiar LAMP detection techniques to detect the LAMP amplification of LIU with familiar results with a reasonable expectation of success.
LIU does not explicitly teach more than one LNA on 5’-third of primer, to use metallochromic indicator such as PAR, or “regulator for melting temperature,” or 5-Br-PAPS.
As to another PNA, this was a known option, based on LIU, to further affect primer affinity (para. 0043). A skilled artisan would have been motivated to optimize the primer with another PNA, similar to AceIN-B3bL, in the 5’-third of the primer based on the specific application.
As to metallochromic indicator such as PAR, or “regulator for melting temperature,” these techniques were very familiar for LAMP detection. For example, TANNER teaches
Kits and methods are provided for performing multiplex Loop-Mediated Isothermal Amplification (LAMP) reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting the viral nucleic acid target
(Abstract). Metallochromic indicator dyes such as 4-(2-pyridylazo) resorcinol (PAR) are well-known option in LAMP to detect reactions without the need for expensive detection equipment (paras. 0002, 0007). Furthermore, using SSB, TCEP and guanidine hydrochloride in the LAMP reaction were very familiar options (paras. 0043, 0098-0103). A skilled artisan would have been motivated to include these familiar LAMP detection solutions in the LAMP reaction of LIU to achieve similar increase in efficiency with a reasonable expectation of success.
Finally, 5-Br-PAPS chelating coloring agent was a known substitute for PAR chelating coloring agent that performed the same function:
Examples of chelating coloring agents include 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-3-sulfopropylamino)phenol (sometimes abbreviated as 5-Br-PAPS hereinafter), 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-3-sulfopropylamino)phenol (sometimes abbreviated as nitro-PAPS hereinafter), 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-3-sulfopropylamino)aniline (sometimes abbreviated as 5-Br-PSAA hereinafter), Bismuthiol II, BPA, Calcein, Calcein Blue, chromotropic acid, Cu-PAN, diantipyrylmethane, murexide, nitroso-PSAP, o-phenanthroline, PAN, PAR, PDTS, PR, SATP, Tiron, TPPS, TPTZ, XO or Zincon
(HIGURASHI, para. 0051).
Thus, the claims are obvious.
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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/AARON A PRIEST/Primary Examiner, Art Unit 1681
