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 .
Priority
This application claims priority to provisional application 63/357,843. Therefore, it is entitled to the 1st Jul 2022 priority date of the parent application.
Amendments and Arguments
This action is in response to papers filed 29th June 2026 in which claims 1-2 and 24-25 were amended, claims 4, 8, 13, 18-21 and 28 were previously canceled, and no new claims were added. All of the amendments have been thoroughly reviewed and entered.
Any rejection or objection not reiterated herein has been overcome by amendment.
Applicant's arguments regarding the §103 rejections have been fully considered but are
unpersuasive. The §103 rejections are maintained and rewritten for clarity. These have been addressed below the maintained §103 rejections.
Status of Claims
Claims 1-3, 5-7, 9-12, 14-17,and 22-27 are under consideration.
Maintained Claim Interpretation
Claim 1 is being interpreted as a method of producing a transcribed RNA product comprising the following steps:
a. transcription reaction wherein a yield of 1 g to 25 g of single stranded transcribed RNA per liter of liquid of the transcription reaction prior to the addition of DNase in step b. is produced.
b. stopping the transcription reaction
The transcription reaction mixture of a. comprises:
a buffer solution comprising Mg2+, molar concentration of Mg2+ is 2 to 15 mM above the total molar concentration of all rNTPs plus the optional RNA capping reagent,
linear DNA (L.DNA) template,
ribonucleoside tri-phosphate (rNTPs),
optionally an RNA capping reagent,
and RNA polymerase; wherein RNA polymerase/L. DNA template mass ratio is between 0.25 and 3;
b. The stopping transcription reaction comprises:
deoxyribonuclease (DNase) or ethylenediaminetetraacetic acid (EDTA).
As seen in the table below, Claim 1 and claim 25 recite same subject matter except for the bolded limitation and the order of steps presented in claim 25.
Claim 1 is being interpreted as a method of producing a transcribed RNA product comprising the following steps: i. transcription reaction, wherein the transcription reaction mixture of i. comprises:
Claim 25 is A method of producing a transcribed RNA product comprising:
a buffer solution comprising Mg2+, molar concentration of Mg2+ is 2 to 15 mM above the total molar concentration of all rNTPs plus the optional RNA capping reagent
a. loading a reactor vessel with a buffer solution comprising Mg2+ and nuclease free water, molar concentration of Mg2+ is 2 to 15 mM above the total molar concentration
of all rNTPs plus the optional RNA cap after steps (a)-(b) are completed
linear DNA (L.DNA) template
b. adding a solution comprising L.DNA template spiked with 200-1000 mM NaCl;
ribonucleoside tri-phosphate (rNTPs)
c. adding rNTPs and optionally RNA cap
RNA polymerase; wherein RNA polymerase/L. DNA template mass ratio is between 0.25 and 3;
d. adding RNA polymerase and mixing the resulting transcription solution wherein the RNA polymerase/DNA template mass ratio is between 0.25 and 3; and,
yield of 1 g to 25 g of single stranded transcribed RNA per liter of liquid of the transcription reaction prior to the addition of DNase in step (ii) is produced.
1 to 25 g of
single stranded transcribed RNA/ L of liquid of the transcription reaction prior to the addition of
DNase in step (b) is produced.
ii. The stopping transcription reaction comprises:
deoxyribonuclease (DNase) or ethylenediaminetetraacetic acid (EDTA).
e. adding DNase to the transcription solution;
Claims 24 and 27 are being interpreted as methods wherein when yields of greater than 5 g/ L of RNA transcripts occur, these are accompanied by reduced amounts of dsRNA impurities.
Maintained 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:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. 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.
Claim(s) 1, 3, 5-7, 9-12, 14, 17, and 22-24 remain rejected under 35 U.S.C. 103 as being obvious over Dias (US 2018/0258423 A1, IDS) in view of Piao (WO 2021/158789 A1) as made in Non-Final Office Action dated 3/30/2026 and reiterated below.
This rejection includes one new sentence elaborating on the teachings of Piao. See this new sentence bolded and introduced within Piao’s teachings.
Regarding claim 1, Dias teaches methods of producing large scale RNA in a buffered transcription reaction mix using RNA polymerase (whole document). In an embodiment with T7 polymerase, 10g of CFTR mRNA is prepared([0200] a 10 gram batch of CFTR mRNA; Fig. 24B). See two pertinent citations that read on instant claim 1 limitations:
[0165]: The T7 transcription reaction consisted of 1 xT7
transcription buffer (80 mM HEPES pH 8.0, 2 mM Spermidine,
and 25 mM MgCl2 with a final pH of7.7), 10 mM
DTT, 7.25 mM each ATP, GTP, CTP, and UTP, RNAse
Inhibitor, Pyrophosphatase, and T7 Polymerase.
a) Scaled up reaction [0166]: 20 mg of a linearized double stranded
DNA plasmid with an RNA polymerase-specific promoter,
10 mg RNA polymerase, RNase inhibitor, pyrophosphatase,
5 mM NTPs, 10 mM DTT and a reaction buffer (l0x-250
mM Tris-HCI, pH 7.5, 20 mM spirmidine, 50 mM NaCl,)
was used and quantity sufficient (QS) to 200 ml with
RNase-free water then incubated at 37 C for 60 min.
The amounts in [0166] are for each gm of mRNA produced. Therefore, if 1gm is produced in 200mL, when an yield of 10gm is obtained as in [0200], this must be 10g/2000mL, which is 5g/L, which is within the recited range of 1g/L to 25g/L.
As seen in the citation, ratio of DNA to polymerase is 20:10. Therefore, ratio of polymerase to DNA is 1:2, which is 0.5; i.e., within the required ratio of 0.25 to 3.
Regarding claim 3, Dias teaches wherein the transcription reaction mixture of step (a) further comprises one or more of the group consisting of RNase inhibitor and inorganic pyrophosphatase [0167].
Regarding claim 5, Dias teaches wherein the temperature during step a) and b) is in a range from about 30 °C to about 40 °C [0167].
Regarding claim 6, Dias teaches wherein the L.DNA template is from about 0.01 mg/mL to about 0.3 mg/mL in the transcription reaction mixture [0167]. As seen in the citation, DNA is 20mg/200mL, which is 1mg/10mL, which is 0.1mg/1mL.
Regarding claim 7, Dias teaches wherein the RNA polymerase is T7 polymerase [0165].
Regarding claim 9, Dias teaches wherein the transcription reaction mixture of step (a) is allowed to react for at least 20 minutes prior to step (b) stopping the transcription reaction [0167].
Regarding claim 10, Dias teaches the Cap and Tail (C/7') Reaction is carried out on purified DNA [0167].
Regarding claim 12, Dias teaches wherein the RNA polymerase is 0.0125 to 0.15 μg/μL T7 polymerase [0167]. As seen in the citation, T7 polymerase is 10mg/200mL, which is 0.05μg/1 μL.
Regarding claim 14, Dias teaches wherein the amount of single stranded transcribed RNA is measured after purification of the transcription mixture after step (b) via a silica column (the purification via silica column various ways to purify, [0120]; and Qiagen RNA maxi column, [0165]; 1 to 2 μg of RNA was treated…[0170]). Since Dias runs 1-2 μg of purified RNA on a gel, a measurement has to be done to further treat 1-2 μg.
Regarding claim 22, Dias teaches wherein the L.DNA template comprises an open reading frame that encodes a vaccine antigen, enzyme, antibody, receptor, tRNA, and/ or a protein (antibody, para [0150]-[0151]; endonuclease, para [0153]; CFTR mRNA, [0200]).
Regarding claim 23, Dias teaches wherein the total concentration of rNTPs is at least 8 mM (5 mM NTPs, [0167]). If 5 mM of each NTPs are used, then for four of the NTPs, this would amount to 20mM. Since the MgCl2 has not been scaled up, the MgCl2 remains at 25mM. 25mM is greater than the total of all rNTPs.
Dias is silent on whether Mg2+ is carried forward in the large scale reaction, as required by instant claims 1 and 11.
Dias lacks a teaching on a solvent from the recited group in claim 17.
However, before the effective filing date of instant invention, Piao had taught a method of producing a transcribed RNA product comprising reacting a transcription reaction mixture comprising a RNA polymerase, resulting in increased yield and lacking dsRNA impurities (abstract). Piao taught wherein the reaction mixture in the method of producing a transcribed RNA product contains Mg2+ ([0089]: Magnesium/magnesium ions and DTT (or some other reducing agent) are often included in transcription buffers). Piao further taught “Magnesium in the transcription mixture is, in certain embodiments, at a final concentration of about 12 to about 60 mM” [0089]. Stock Magnesium in Piao’s transcription reaction mixture is at 165 mM Mg2+ (The starting reaction mixture comprises, in certain embodiments, 50 ng/μL DNA plasmid template, rNTPs (5 mM), CleanCap AG (4 mM), reaction buffer (l0X buffer: 400 mM HEPES (pH 7.2-7.5), 100 mM DTT, 20 mM spermidine, 0.02% triton X-100, 165 mM Mg2+), T7 RNA polymerase (about 4000 U/mL), RNase inhibitor (about 1000 U/mL), and inorganic pyrophosphatase (about 2 U/mL), [0090]). The transcribed RNA yields without any additives are at least 2.8 mg/mL (Tables 2 - 14).
Thus, Piao teach that Mg2+ is essential for the transcription reaction when an yield of 1g/L to 25g/L of transcribed RNA is produced. The recited molar concentration of Mg2+ is 5 to 15 mM above the total molar concentration of all rNTPS plus the molar concentration of any optional RNA capping reagent: In Piao’s reaction, if 5 mM of each NTPs are used, then for four of the NTPs, this would amount to 20mM, plus 4 mM of the capping reagent = 24mM total. To be 2 to 15 mM and 5 to 15 mM above this total molar concentration, as required by claims 1 and 11 respectively, one would require a minimum of 26mM and 29mM respectively. Piao’s MgCl2 in at least some embodiments is greater than this required molarity.
Regarding claim 17, Piao had taught a method of producing a transcribed RNA product comprising reacting a transcription reaction mixture comprising a RNA polymerase, wherein the method comprises adding a solvent (chaotropic agent) such as EtOH to the transcription reaction mixture which acts to reduce the formation of double-stranded ribonucleic acid (dsRNA) during in vitro transcription (claims 1-6 A method of reducing, minimizing, or inhibiting the formation of double-stranded ribonucleic acid (dsRNA) during in vitro transcription, comprising adding at least one chaotropic agent to an in vitro transcription reaction mixture ... wherein the transcription yields ribonucleic acid (RNA) ... wherein the at least one chaotropic agent is selected from the group consisting of urea, formamide, sodium salicylate, ethanol, sodium perchlorate, arginine, n-butanol, thiourea, and 2-propanol). Piao teaches the composition comprising EtOH in a concentration of 1 to 10 % v/v (claim 18 wherein the ethanol is at a concentration of from about 0.4M to less than about 1.6M). Piao teaches addition of the solvent maintains mRNA yield while reducing the formation of unwanted dsRNA during in vitro transcription (claims 1, 5 wherein the amount or yield of RNA is not significantly reduced by the addition of the at least one chaotropic agent).
It would have been obvious to one of ordinary skill, in the art at the time, to modify the method of Dias by scaling up Mg2+ ions and including at least one chaotropic agent as taught by Piao to gain the advantage of reducing the formation of unwanted dsRNA during in vitro transcription. Further, it would have been obvious to one of ordinary skill in the art to have applied the transcription reaction mixture components taught by Piao to the mRNA transcription methods taught by Dias, in order to improve the overall quality of the mRNA transcription products generated. One would have had reasonable expectation of success in doing so because both references taught methods of improving yield of IVT RNA. See MPEP 2143 I (A) and 2144 II.
Regarding claim 24, the method of producing a transcribed RNA product comprising reacting a transcription reaction mixture comprising a RNA polymerase, wherein the method comprises adding a solvent (chaotropic agent) such as EtOH to the transcription reaction mixture which acts to reduce the formation of double-stranded ribonucleic acid (dsRNA) during in vitro transcription (claims 1-6), wherein a yield of about 5 g/L of RNA transcript results in a reduced amount of dsRNA as compared to a transcription reaction wherein the yield is less than about 5 g/L (para [0121]-[0122]).
Thus, Dias in view of Piao make obvious instant claims 1, 3, 5-7, 9-12, 14, 17, and 22-24.
Claims 2, 15-16, 25 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Dias (US 2018/0258423 A1, IDS) in view of Piao (WO 2021/158789 A1) as applied to claims 1, 3, 5-7, 9-12, 14, 17, and 22-24 above in view of Cavac (Cavac et al., J. Biol. Chem. (2021) 297(3) 100999). Claims 15-16 are evidenced by Martins (R. Martins et al. / J. Chromatogr. A 1355 (2014) 1–14).
Regarding claim 2, the method of claim 1 is discussed above. Neither Dias nor Piao teach wherein the L.DNA template is in a solution comprising 50 mM to 1200 mM NaCl to produce a salt-spiked L.DNA template prior to introduction to the transcription reaction mixture.
However, before the effective filing date of instant invention, Cavac had taught a method of producing transcribed RNA comprising reacting T7 RNA polymerase with linear DNA in the presence of high salt to generate increased yields of highly pure RNA (title, abstract). The taught method results in significantly lower yields of dsRNA impurities (abstract). Regarding amounts of added NaCl, Cavac teach at pg. 3, l col, “At 0.3 M added NaCl, most of the primer extension activity is inhibited, leading directly to an increase in the encoded RNA yield.”; at pg. 6, r col, 1st para “a practical optimum of about 0.3 M added NaCl provides a good trade-off of purity versus yield”. Also see Fig. 2. Cavac further teach the concentrations of higher salt will vary with the needs of the user (For practical consideration, users can determine the optimal concentration of added salt, depending on their RNA sequence, targeted degree of purity, and desired yield, at pg. 6, r col, 3rd para).
It would have been obvious to one of ordinary skill, in the art at the time, to modify the method of Dias and Piao, to include a higher concentration of salt in the reaction mix, for the advantage of obtaining high purity yield as demonstrated by Cavac. With respect to the order of steps, this is a matter of design choice. See MPEP 2144.05 II and 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."). With respect to the order of steps, in particular, it is noted that the courts have held that 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); Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959)). See MPEP §2144.04 IV C.
Therefore, the claimed order of steps of spiking the stock template DNA solution or incorporating the high salt in the reaction mix, is an obvious variant of the steps of the cited prior art.
Regarding claims 15 and 16, Cavac teach in vitro T7 RNA polymerase transcription is often followed by extensive purification methods (abstract). Cavac teach reliance on different purification methods depends upon the length of RNA obtained (At long RNA lengths, purification methods do not have the resolution to eliminate all impurities (for example, separating a 2000 base transcript from a 2040 base impurity) and purifications lead to a loss in yield, (pg. 1, r col, 2nd para). As evidenced by Martins cited by Cavac at pg. 1, r col, 2nd para, one such method is affinity purification. Martin further evidences, more than one purification step is required to achieve higher RNA enrichments (pg. 9, l col, first line). Martin further evidences, that the major advantage of these systems is the broad applicability to any RNA of interest, even though limited to the need of several design issues, which may lead to longer optimization processes. Thus, Cavac teaches that the choice of purification and order of use is a results-effective variable that is readily optimized by one of ordinary skill in the art as needed or desired.
It would have been obvious to one of ordinary skill in the art apprised of the teachings of Dias and Piao to also come across the teachings of Cavac and incorporate an affinity purification method because Cavac indicates that extensive purification is often required and many methods exist for the same, and the choice of which method to pick depends upon the RNA that needs purification. Therefore, for any given IVT procedure, the skilled artisan would recognize that the purification methods to follow are subject to optimization via routine methods. One would have had reasonable expectation of success in optimizing an affinity purification method after silica column purification to obtain a highly pure IVT RNA, as these were well-known methods, well-within the grasp of the ordinary skilled artisan. See MPEP §2143 A and 2144.05 II.
Regarding claim 25, as discussed in claim interpretation, the method recited in claim 25 is substantially identical to the method recited in claim 1, except for the limitation of L.DNA in salt solution. Therefore, the discussion of claim 1 in the §103 rejection in view of Dias and Piao and the discussion of claim 2 in the §103 rejection in view of Cavac is incorporated herein.
Regarding claim 27 that depends upon claim 25, Cavac further teach double-stranded impurities are significantly reduced when a “high-salt transcription” method is followed (abstract). This reads on the limitation of claim 24, wherein a yield of greater than about 5 g/ L of RNA transcript results in a reduced amount of dsRNA as compared to a reaction which is not carried out in high-salt concentration.
It would have been obvious to one of ordinary skill, in the art at the time, to modify the method of Dias and Piao, to include a higher concentration of salt in the reaction mix, for the advantage of obtaining high purity yield as demonstrated by Cavac. One would have had reasonable expectation of success in optimizing a high-salt step, as Cavac provides a detailed methodology to do so. See MPEP §2143 A and 2144.05 II.
Regarding claim 26, the discussion of claim 1 and 17 in the §103 rejection in view of Dias and Piao, the discussion of claim 2 in the §103 rejection in view of Cavac, and the rationale for combining references are incorporated herein.
The combination of the limitations taught by Dias, Piao, and Cavac put together will result in the limitations of claim 26. With respect to the order of steps, again, it is noted that the courts have held that 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); Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959)). See MPEP §2144.04 IV C.
Thus, Dias and Piao in view of Cavac make obvious instant claims 2, 15-16, and 25 - 27.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Response to Arguments
Applicant's arguments filed 6/29/2026 regarding §103 rejections have been fully considered but they are not persuasive.
Applicants argue on pg. 4, last para,
“"Magnesium in Piao's transcription reaction mixture is at 165 mM Mg2+" (Office Action, pg. 12-13). However, the passage of Piao cited for this proposition does not support the Office's conclusion. As recited in the Office Action itself, the feature of "165 mM Mg2+" is the concentration in a "10x buffer".”. Then, Applicants go on to state: A person of ordinary skill in the art would understand that the concentration of a "10x buffer" does not reflect the concentration of reagents in a single ("lx") reaction.
Then bridging pgs. 4 and 5, Applicants state, paragraph [0097] of Piao, "The standard IVT recipe consisted of: 5 mM of each NTP, 50 ng/pL plasmid, 16.5 mM Mg2+ .. ." (emphasis added). This concentration is less than the "24mM total" concentration of dNTPs plus capping reagent cited by the Office (Office Action, pg. 13). Therefore, Piao fails to provide the missing teaching, gives no reason to use a higher concentration of Mg2+ within the scope of the present claims, and provides no basis for reasonably predicting the results thereof (much less any technical advantage).”
Applicants arguments are unpersuasive because:
the passage of Piao cited is [0089]. This same passage includes a sentence that states an embodiment wherein the Mg2+ concentration is within the instant requirement. See this new sentence in instant rejection.
paragraph [0097] of Piao is “The standard IVT recipe”, as Applicants have correctly stated.
Regarding diluting the 10X buffer to 1X, such dilution has been done in Piao’s standard IVT recipe. One of skill in the art knows that stock buffers are made concentrated, i.e., 10X, so that dilution to any lower concentration may be done as required by the reaction conditions. A dilution to 1x is not the only possibility or requirement.
Therefore, as an embodiment of Piao shows, a higher than 16.5mM Mg concentration may be obtained.
Applicants have not provided any arguments rendering the other references invalid for the reasons the art was applied nor have they provided any evidence showing there was no reasonable expectation of success.
Applicants arguments are not dispositive. The §103 rejection is maintained.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. 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.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SHABANA MEYERING, Ph.D. whose telephone number is (703)756-4603. The examiner can normally be reached M - F: 9am to 5pm EST.
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SHABANA S. MEYERING, Ph.D.
Examiner
Art Unit 1635
/SHABANA S MEYERING/ Examiner, Art Unit 1635 /RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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