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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 December 19, 2025 has been entered.
DETAILED ACTION
3. Claims 1, 3, 6, 8 – 19, and 20 – 22 are pending in this application. Applicant’s Amendment and Remarks, filed December 19, 2025, is entered, wherein claim 1 is amended, claims 16 – 22 are withdrawn, and claims 2, 4 – 5, and 7 are canceled.
Therefore, claims 1, 3, 6, and 8 – 15 are currently examined.
Priority
This application is a national stage application of PCT/US2020/030112, filed April 27, 2020, which claims benefit of domestic application 62/838,852, filed April 25, 2019.
Withdrawn Objections
5. The objection of claim 1 in the previous Office Action, mailed September 25, 2025, is withdrawn in view of the amended claim 1.
Withdrawn Rejections
6. The rejection of claims 1, 8 – 10, 12, and 15 in the previous Office Action, mailed September 25, 2025, under 35 U.S.C. 103 as being unpatentable over Shen et al. in view of Johnson et al. has been considered and is withdrawn in view of the amended claim 1.
The rejection of claims 3 and 6 in the previous Office Action, mailed September 25, 2025, under 35 U.S.C. 103 as being unpatentable over Shen et al. in view of Johnson et al., and further in view of Bakaltcheva et al. has been considered and is withdrawn in view of the amended claim 1.
The rejection of claims 11 and 13 – 14 in the previous Office Action, mailed September 25, 2025, under 35 U.S.C. 103 as being unpatentable over Shen et al. in view of Johnson et al., and further in view of Iwata et al., Yang, Heili et al., and Thermo Scientific has been considered and is withdrawn in view of the amended claim 1.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
i. Determining the scope and contents of the prior art.
ii. Ascertaining the differences between the prior art and the claims at issue.
iii. Resolving the level of ordinary skill in the pertinent art.
iv. Considering objective evidence present in the application indicating obviousness or
nonobviousness.
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.
Claims 1, 8 – 10, 12, and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Shen et al. (US5556771) in view of Al-Hussein (Journal of Pharmaceutical Sciences, 2012, Vol. 101, Issue 7, page 2534 – 2544, Reference included with PTO-892).
a. Regarding claims 1, 8 – 10, 12, and 15, Shen et al. teach the invention that is directed to stabilized reverse transcriptase and RNA polymerase compositions for use in nucleic acid amplification. The compositions and kits comprising dried formulation of reverse transcriptase and RNA polymerase able to be stored at ambient temperature for prolonged periods of time without substantial losses of enzymatic activities (Col. 6, lines 9 – 13). In example 7, sucrose at a concentration of 0.2 M is used as a cryoprotectant stabilizing agent in the lyophilization of reverse transcriptase and the stabilizing effect of sucrose appears to be good (Col. 21, lines 24 – 28). Shen et al. further teach that lyophilization may also be done in the presence of mannitol (Col. 4, lines 60 – 61). Furthermore, Shen et al. disclose that one method for nucleic acid amplification includes incorporating a promotor sequence into one of the primers using in the PCR reaction and then, after amplification by the PCR method, using the double-stranded DNA as a template for the transcription of single-stranded RNA by a DNA directed RNA polymerase (Col. 2, lines 43 – 51). The invention is directed to single containers comprising dried formulations containing MMLV-RT and T7 RNA polymerase, one or more cryoprotectant excipients comprising either or both trehalose and PVP, nucleotide triphosphates, and metal ions and co-factors necessary for said enzymatic activities wherein, upon reconstitution of the stabilized lyophilizate and addition of a target nucleic acid and one or more appropriate primers, the formulation is in a convenient and cost-effective form for nucleic acid amplification without the need for excessive handling (Col. 6, lines 22 – 34). In one example, a buffer containing 20 mM HEPES, 5 mM NALC, 0.1 mM EDTA, 0.1 mM zinc acetate, 0.2 % TRITON x-102, and 0.2 M trehalose is used (Col. 13, lines 62 – 64).
However, Shen et al. do not teach the combination use of sucrose and mannitol as well as the concentration of mannitol.
Al-Hussein discloses the effect of mannitol crystallization in mannitol-sucrose systems on lactate dehydrogenase (LDH) stability during freeze-drying (Title). Protein formulations with different weight ratios of mannitol to sucrose are being tested. Protein stability is then evaluated both functionally by measuring the activity recovery of LDH after free-drying and structurally by analyzing the protein secondary structure (Abstract). The formulation used in the study are shown (page 2536, Left Col., Table 1):
PNG
media_image1.png
243
346
media_image1.png
Greyscale
.
The activity recovery of different freeze-dried LDH formulations are shown (page 2537, Right Col. Figure 1):
PNG
media_image2.png
322
339
media_image2.png
Greyscale
.
For the formulations containing relatively high sucrose concentrations, no significant differences in LDH activity could be observed between mixtures (page 2538, Left Col., para. 1).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the composition as taught by Shen et al. with both sucrose and mannitol in view of Al-Hussein because Al-Hussein test the protein stability in mannitol-sucrose systems after lyophilization of protein and Al-Hussein specifically discloses formulations of protein with different weight ratios of mannitol to sucrose. Figure 1 shows that protein activity in formulations, M50/S50, M30/S70, M10/S90, remain high after freeze-drying. One would have been motivated to combine the composition as taught by Shen et al. with both sucrose and mannitol in view of Al-Hussein and considered the amount of mannitol and sucrose used for lyophilization based on Al-Hussein because Al-Hussein teaches that the combination of mannitol and sucrose will stabilize protein. The concentration of mannitol from 5 mg/mL to 50 mg/mL is calculated as between 27.4 mM and 274.0 mM and the concentration of sucrose from 5 mg/mL to 50 mg/mL is between 14.6 mM and 146.1 mM. In one formulation M30/S70, the concentrations of mannitol and sucrose are 82.3 mM and 102.2 mM, respectively. The protein recovery remains high at this concentration. Based on the data, one would have performed routine experimentation to discover the best concentration of mannitol for achieve the optimal protein stability. Moreover, there is no teaching regarding the composition further comprises glycerol and organic solvent. It is expected that glycerol and organic solvent are absent in the composition. Finally, it would have been obvious for a skilled artisan to rehydrate the lyophilized composition with water to allow RNA transcription takes place. Therefore, one of the skills in the art would have had a reasonable expectation of success to combine the composition as taught by Shen et al. with both sucrose and mannitol in view of Al-Hussein as Shen et al. teach the lyophilized composition comprising T7 RNA polymerase, target nucleic acid sequence, nucleotide triphosphates, buffering agent, sucrose, and mannitol and Al-Hussein teaches that sucrose and D-mannitol have been used as excipients for the lyophilization of proteins, thereby, the combination of sucrose and D-mannitol will yield predictable results.
Claims 3 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Shen et al. (US5556771) in view of Al-Hussein (Journal of Pharmaceutical Sciences, 2012, Vol. 101, Issue 7, page 2534 – 2544, Reference included with PTO-892) as applied to claims 1, 8 – 10, 12, and 15 above, and further in view of Bakaltcheva et al. (Thrombosis Research, 2007, Vol. 120, Issue 1, page 105 – 116, cited in the previous Office Action mailed September 25, 2025).
b. Regarding claims 3 and 6, Shen et al. and Johnson et al. teach the limitations discussed above.
However, these references do not teach the composition further comprising trehalose or sorbitol.
Bakaltcheva et al. teach that the two disaccharides sucrose and trehalose are among the most commonly used protein stabilizers in freeze-dried formulations. Their protective properties are well-documented, including their ability to protect coagulation factor proteins and fibrinogen. Sucrose, trehalose, raffinose and arginine are listed as stabilizing agents (page 107, Left Col., para. 3). Bulking agents are compounds that tend to crystalline upon the freezing stage of the freeze-drying process, and in doing so, provide the freeze-dried cake a pharmaceutically elegant (i.e. noncollapsed) structure, wherein mannitol, sorbitol are among the commonly used bulking agents (page 107, Left Col., para. 4).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the composition as taught by Shen et al. with trehalose and/or sorbitol in view of Bakaltcheva et al. because Bakaltcheva et al. teach that trehalose and sorbitol are commonly used in the freeze-drying process for the stability and bulking effect. It would have been obvious for one of ordinary skill in the art to do this because both trehalose and sorbitol are known in the art for the purpose of freeze-drying proteins. One of ordinary skill in the art would have had a reasonable expectation of success to combine the composition as taught by Shen et al. with trehalose and/or sorbitol in view of Bakaltcheva et al. because it is well known to combine different components to yield the high versatility for freeze-drying different proteins.
Claims 11 and 13 – 14 are rejected under 35 U.S.C. 103 as being unpatentable over Shen et al. (US5556771) in view of Al-Hussein (Journal of Pharmaceutical Sciences, 2012, Vol. 101, Issue 7, page 2534 – 2544, Reference included with PTO-892) as applied to claims 1, 8 – 10, 12, and 15 above, and further in view of Iwata et al. (Bioorganic & Medicinal Chemistry, 2000, Vol. 8, Issue 8, page 2185 – 2194, cited in the previous Office Action mailed September 25, 2025), Yang (Journal of Visualized Experiments, 2016, Issue 115, cited in the previous Office Action mailed September 25, 2025), Heili et al. (Journal of Chemical Education, 2018, Vol. 95, Issue 10, page 1867 – 1871, cited in the previous Office Action mailed September 25, 2025), and Thermo Scientific (Bovine Serum Albumin, 2017, cited in the previous Office Action mailed September 25, 2025).
c. Regarding claims 11 and 13 – 14, Shen et al. and Johnson et al. teach the limitations discussed above. Shen et al. further teach a reconstitutable dried formulation comprising a reverse transcriptase, an RNA polymerase, ribonucleotide triphosphates, deoxyribonucleotide triphosphates, zinc and/or magnesium salts, and a reducing agent in a single container (Col. 7, lines 35 – 40).
However, these references do not teach the composition further comprises a polyamine, an RNase inhibitor, inorganic pyrophosphatase, bovine serum albumin, and purified transcription factors. These references also do not teach the lyophilized composition comprising the DNA transcription template that encodes an aptamer and further comprises a dye that fluoresces.
Iwata et al. teach that T7 RNA polymerase is activated by the specific binding of the polyamine additive to produce RNA transcripts with fidelity to the template DNA (Abstract).
Yang teaches that in vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression, including elongation factors such as NusA that regulate transcriptional pausing (Abstract). Except for elongation, transcription factors are also responsible for the regulation of RNAP activities during termination phases (page 1, para. 1). Purified NusA is used in the transcription reaction (page 3, para. 1). Yang also teaches that high quality RNase inhibitor must be incorporated in the transcription reaction mixture for preventing the degradation of the transcription product (page 5, para. 3).
Heili et al. teach the main experiment that involves the transcription of a Broccoli aptamer from a DNA template using T7 RNA polymerase. In vitro transcription reactions with T7 RNA polymerase require at minimum: (1) a DNA template coding for the RNA of interest, with a double-stranded promoter region for T7 RNA polymerase, (2) T7 RNA polymerase, (3) r(NTPs); and (4) a suitable buffer. The transcription reactions contain these components, as well as (5) DFHBI-1T, the small-molecule ligand that becomes fluorescent upon binding to the transcribed aptamer, and inorganic pyrophosphatase (page 1868, Right Col., para. 3). Inorganic pyrophosphatase hydrolyzes the pyrophosphate leaving group produced by incorporation of an NTP into a nascent strand of RNA, producing two phosphate ions. The inclusion of this enzyme in the transcription mixture prevents the buildup of pyrophosphate, which otherwise would chelate Mg2+ ions that are required for both transcription and folding of the produced aptamer (page 1868, Right Col., para. 4).
Thermo Scientific teaches that bovine serum albumin (BSA) is used for stabilization of enzymes during storage and for enzymatic reactions where the absence of nucleases is essential. BSA increases PCR yields from low purity templates. It also prevents adhesion of enzymes to the reaction tubes and tip surfaces (page 1, Right Col., para. 1).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the composition as taught by Shen et al. with polyamine, an RNase inhibitor, inorganic pyrophosphatase, bovine serum albumin, and purified transcription factors in view of Iwata et al., Yang, Heili et al., and Thermo Scientific because Iwata et al. teach that polyamine is used to activate T7 RNA polymerase, Yang teaches that purified transcription factors will help controlling the transcription reaction and RNase inhibitor helps preventing the degradation of the transcription product, Heili et al. teach that inorganic pyrophosphatase prevents the buildup of pyrophosphate, and Thermo Scientific teaches that BSA is used for stabilization of enzymes during storage. One would have been motivated to combine the composition as taught by Shen et al. with polyamine, an RNase inhibitor, inorganic pyrophosphatase, bovine serum albumin, and purified transcription factors in view of Iwata et al., Yang, Heili et al., and Thermo Scientific because of the benefits disclosed by Iwata et al., Yang, Heili et al., and Thermo Scientific and said combination will yield a composition with improved properties. It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date to substitute the target nucleic acid as taught by Shen et al. with a DNA transcription template encoding aptamer in view of Heili et al. because Heili et al. demonstrate that transcription from such a template predictably yields the same aptamer RNA capable of binding a fluorescent dye. This substitution would represent no more than the predictable use of a known equivalent means to generate the aptamer in vitro. Therefore, one of the ordinary skill in the art would have had a reasonable expectation of success to combine the composition as taught by Shen et al. with polyamine, an RNase inhibitor, inorganic pyrophosphatase, bovine serum albumin, and purified transcription factors in view of Iwata et al., Yang, Heili et al., and Thermo Scientific and to substitute the target nucleic acid as taught by Shen et al. with a DNA transcription template encoding aptamer in view of Heili et al. because it is known in the art for the benefits of combining polyamine, an RNase inhibitor, inorganic pyrophosphatase, bovine serum albumin, and purified transcription factors and it is also known in the art to perform in vitro transcription reaction with DNA transcription template encoding aptamer as disclosed by Heili et al.
Responses to Applicant’s Remarks:
Applicant’s Remarks, filed December 19, 2025, have been fully considered and are found to be not persuasive.
Applicant argues that claim 1 has been amended to recite “sucrose at a concentration of at least about 200 mM and D-mannitol at a concentration of at least about 200 mM” and that the cited references, alone or in combination, fail to teach or suggest a lyophilized composition having these features because Shen et al. do not teach the combined used of sucrose and mannitol and the remaining references fail to remedy this deficiency. Applicant points to Johnson et al. and stated that Johnson et al. teach the ratio of mannitol to sucrose is 2.3 or above to prevent the cake from collapsing during freeze drying. Therefore, a skilled artisan would have expected that the low D-mannitol to sucrose ratio would result in total collapse of the cake. However, the arguments are not persuasive because the amended claim 1 recites “sucrose at a concentration of at least about 200 mM and D-mannitol at a concentration of at least about 200 mM” does not limit sucrose to mannitol to be 1:1 only. Moreover, the new rejection is over Shen et al. in view of Al-Hussein, which demonstrates that low mannitol would not affect protein stability. Johnson et al. is no longer included in the new rejection. Therefore, the argument regarding Johnson et al. is moot.
Conclusion
No claim is found to be allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to HOI YAN LEE whose telephone number is 571-270-0265. The examiner can normally be reached Monday - Thursday 7:30 - 17:30.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, SCARLETT GOON can be reached at 571-270-5241. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/H.Y.L./Examiner, Art Unit 1693
/SCARLETT Y GOON/Supervisory Patent Examiner, Art Unit 1693