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 .
Application Status
The action is written in response to applicant’s correspondence received on 6/10/2024. Claims 1-2, 6-8, 10, 14, 18, 20, 22, 26, 30-31, 33-35, 37, 41-43, and 45 are currently pending in the instant application.
Priority
The instant application claims priority to US Provisional Application 63/190,060, filed on 5/18/2021.
Specification
The use of the term Terminator™, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 18 and 41 are objected to because of the following informalities: Both claims 18 and 41 recites “an amount between 0.012U an 0.1 U per 1 ug of polyribonucleotides, where “an” should be changed to “and”.
Appropriate correction is required.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 26, 31, 33-35, 37, 43 and 45 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Goldberg et al. (US 2018/0169146 A1, published 6/21/2018).
Regarding claim 26, Goldberg teaches a method of circularizing a nucleic acid, comprising taking nucleic acid and ligating the 5’ terminus of the nucleic acid to its 3’ terminus (see paragraph 0022). In a further embodiment, non-circularized (or linear) nucleic acid molecules may be digested with an RNase, e.g. RNase R, Exonuclease T, or XRN-1, preferable the RNase is RNase R and/or XRN-1 (see paragraph 0022). In Fig. 3A, Goldberg teaches a population of circularized and linear mRNA digested with Xrn-1 alone. Absent evidence to the contrary, Goldberg shows the process of RNA digestion can occur with a 5’ exonuclease first, or a 3’ exonuclease first, or together, making the step process of digestion by 5’ first before 3’ moot, if the reaction occurs in a single mixture. Non-circularized nucleic acid molecules may be digested with an RNase after the initial ligation or after the ligation is repeated at least one additional time (see paragraph 0022), leading to an enriched population of circular polyribonucleotides. Furthermore, Goldberg teaches, in a working example, any remaining linear RNA in the circularization reactions was removed using the exonuclease Xrn-1 (NEB, #M0338L), where shown below, the reaction buffer for Xrn-1 according to New England Biolabs directions include Mg2+ in the form of 10mM MgCl2 and 1mM DTT (see paragraph 0129). Goldberg prepares reaction assays according to manufacturer’s recommendation.
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Goldberg also teaches where RNase R and Xrn-1 exonucleases results in the highest purity of circularized product, when combined together (see paragraph 0145). Here, Goldberg satisfies the limitations of claim 26 by disclosing a method where a combined population of circularized and linear mRNA are combined with a 5’ exonuclease (Xrn-1) and a 3’ exonuclease (RNase R), in a digestion buffer that contains 1mM DTT, between the claimed range of 0.1 mM and 5mM DTT.
Regarding claim 31, Goldberg teaches where in Fig. 7A, 5ug of linear and circular forms of an NIuc or eGFP mRNA was exposed to 5 units of RNase R for 45 minutes at 37°C (see paragraph 0192), showing that at least a portion of the linear polyribonucleotide is digested with a 3’ exonuclease (RNase R) at 1U per 1ug (Goldberg recites 5U per 5ug).
Regarding claim 33 and 34, Goldberg teaches the use of XRN-1 (see paragraph 0027), which is known in the art to be a 5’ phosphate dependent exonuclease that requires 5’ monophosphate for activity.
Regarding claim 35, Goldberg teaches the use of RNase R (see paragraph 0027) for the digestion of linear RNAs, which is known in the art to be a 3’ exonuclease.
Regarding claim 37, Goldberg teaches where the digestion step was exposed at 37°C for 45 minutes (see paragraph 0192).
Regarding claim 43, Goldberg teaches where in Fig. 7A, 5ug of linear and circular forms of an NIuc or eGFP mRNA was exposed to 5 units of RNase R for 45 minutes at 37°C (see paragraph 0192), showing that at least a portion of the linear polyribonucleotide is digested with a 3’ exonuclease (RNase R) at 1U per 1ug (Goldberg recites 5U per 5ug).
Regarding claim 45, Goldberg teaches Fig. 8, specifically Fig. 8, where Goldberg analyzes the efficiency with which Xrn-1 removes linear mRNA. Though the amount of circular RNA in the total population of polyribonucleotides is not disclosed in Goldberg, claim 45 does not recite any additional active steps of the method claim. Therefore, inherently, the method of purifying a population of circular and linear mRNA would result in a purified population of circular polyribonucleotides wherein the circular polyribonucleotides is between 40 to 95% of the total population
In view of the foregoing, claims 26, 31, 33-35, 37, 43, and 45 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Goldberg.
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.
Claims 1-2, 6-8, 10, 14, 18, 20, 22, 30, 41, and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Goldberg et al. (US 2018/0169146 A1, published 6/21/2018) in view of Xiao et al. (An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3’ ends, Nucleic Acids Research, Volume 47, Issue 16, pgs. 8755-8769, published 9/19/2019), Dodbele et al. (Best practices to ensure robust investigation of circular RNAs: pitfalls and tips, EMBO Rep. Volume 22, Issue 3, Published 2/25/2021), and Athapattu et al. (Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing, Nucleic Acids Research, Volume 49, Issue 7, published 1/28/2021).
Regarding claims 1, Goldberg teaches a method of circularizing a nucleic acid, comprising taking nucleic acid and ligating the 5’ terminus of the nucleic acid to its 3’ terminus (see paragraph 0022). In a further embodiment, non-circularized (or linear) nucleic acid molecules may be digested with an RNase, e.g. RNase R, Exonuclease T, or XRN-1, preferable the RNase is RNase R and/or XRN-1 (see paragraph 0022). In Fig. 3A, Goldberg teaches a population of circularized and linear mRNA digested with Xrn-1 alone. Absent evidence to the contrary, Goldberg shows the process of RNA digestion can occur with a 5’ exonuclease first, or a 3’ exonuclease first, or together, making the step process of digestion by 5’ first before 3’ moot, if the reaction occurs in a single mixture. Non-circularized nucleic acid molecules may be digested with an RNase after the initial ligation or after the ligation is repeated at least one additional time (see paragraph 0022), leading to an enriched population of circular polyribonucleotides. Goldberg also teaches where RNase R and Xrn-1 exonucleases results in the highest purity of circularized product, when combined together (see paragraph 0145). Furthermore, Goldberg teaches where in Fig. 7A, 5ug of linear and circular forms of an NIuc or eGFP mRNA was exposed to 5 units of RNase R (see paragraph 0192), showing that at least a portion of the linear polyribonucleotide is digested with a 3’ exonuclease (RNase R) at 1U per 1ug (Goldberg recites 5U per 5ug).
Here, Goldberg satisfies the limitations of claim 1 which discloses a method where a combined population of circularized and linear mRNA are combined with a 5’ exonuclease (Xrn-1) and a 3’ exonuclease (RNase R) and wherein the 3’ exonuclease is added at an amount between 0.4 U to 4 U.
Regarding claim 2, Goldberg teaches where the 5’ triphosphate of the nucleic acid is converted into a 5’ monophosphate (see paragraph 0022).
Regarding claim 6 and 7, Goldberg teaches where the RNase can be Xrn-1 (see paragraph 0022), which is a known 5’ exonuclease that targets the 5’ monophosphate moiety.
Regarding claim 8, Goldberg teaches there the RNase can be RNase R or Exonuclease T (see paragraph 0022).
Regarding claim 10, Goldberg teaches in a working example where in Fig. 7A, 5ug of linear and circular forms of an NIuc or eGFP mRNA was exposed to 5 units of RNase R for 45 minutes at 37°C (see paragraph 0192).
Regarding claim 14, Goldberg teaches, in a working example, any remaining linear RNA in the circularization reactions was removed using the exonuclease Xrn-1 (NEB, #M0338L), where shown below, the reaction buffer for Xrn-1 according to New England Biolabs directions include Mg2+ in the form of 10mM MgCl2 and 1mM DTT (see paragraph 0129). Goldberg prepares reaction assays according to manufacturer’s recommendation.
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Regarding claim 20, Goldberg teaches where in Fig. 7A, 5ug of linear and circular forms of an NIuc or eGFP mRNA was exposed to 5 units of RNase R for 45 minutes at 37°C (see paragraph 0192), showing that at least a portion of the linear polyribonucleotide is digested with a 3’ exonuclease (RNase R) at 1 U per 1ug (Goldberg recites 5U per 5ug).
Regarding claim 30 and 41, Goldberg teaches the limitations of the method of claim 26, as described above.
Regarding claim 1, 18, 30, and 41, Goldberg does not teach where a portion of the linear polyribonucleotide is digested with 5’ exonuclease in an amount between 0.01 U and 0.2 U per 1ug of polyribonucleotides. Goldberg instead teaches a 5’ exonuclease (Xrn-1) being added at 5 U and 10 U for 500NT or 1000NT RNA (see paragraph 0142 or Fig. 3A and 3B) or where 5 ug of linear or circularized 500 NT-long RNA constructs were incubated for 1 hour with one of the indicated exonucleases (see paragraph 0146).
Regarding claim 14, Goldberg does not teach the digestion buffer comprising Mg2+ or DTT, but does disclose the reaction buffer with the Xrn-1 enzyme is the NEB recommended buffer, which does contain Mg2+ in the form of MgCl2 (see paragraph 0129).
Regarding claim 1 and 18, Xiao teaches where RNase R digests many but not all linear RNAs (see abstract), specifically RNAs with highly structured 3’ ends, including snRNAs and histone mRNAs. Xiao also discloses that RNase R abruptly stalls at internal G-quadruplex structures, thereby only partially degrading linear RNAs that contain them. This was in part due to K+ ions commonly used in RNase R reaction buffers, which stabilize said G-quadruplex structures, and by replacing K+ ions with Li+ ions in the reaction buffer, it was sufficient for RNase R to proceed and fully degrade the linear RNAs (see introduction). Furthermore, Xiao teaches that for even purer population of circular RNAs to be isolated, inclusion of additional exonucleases may be needed. For example, mRNA 5’ cap structures could be removed using RNA 5’ pyrophosphohydrolase (RppH), followed by digestion with the 5’ exonuclease Xrn1. Xiao teaches that by using both Xrn1 and RNase R, it may be possible to degrade almost all linear RNAs to completion, including RNAs with modified nucleotides at their 3’ ends (see discussion).
Xiao also teaches where in the RNase R treatments, 10 U of RNase R was added to the reaction mixture (see section titled ‘Poly(A) tailing and RNase R treatments).
Xiao does not teach the specific concentration of Xrn-1 used.
Regarding claims 1 and 18, Dodbele teaches a method of allowing RNase R to degrade more linear RNAs by treating linear mRNA with a poly(a) polymerase followed by RNase R digestion, however, this protocol still does not remove all linear RNAs. Dodbele further notes that there are differences in digestion efficiency across commercial lots of RNase R, o one should always verify the reaction conditions used in an experiment lead to efficient digestion of housekeeping linear mRNAs. In addition, some circular RNAs (especially larger ones) have been found to be sensitive to RNase R digestion, especially when high RNase R concentration are used, and this may be due to nicking by contaminating endonucleases (see section titled ‘RNase R digests many, but not all linear RNAs).
Dodbele does not teach the use or specific concentration of Xrn-1.
Regarding claim 14, Athapattu teaches that for Xrn-1 activity, the divalent cation Mg2+ acts as a cofactor to carry out its function as an exoribonuclease (see introduction).
It would have been obvious to one with ordinary skill in the art, before the effective filing date, to combine the teachings of Goldberg with the teachings of Xiao, Dodbele, and Athapattu to arrive at a method of producing an enriched population of circular polyribonucleotides by adding 0.01 U to 0.2 U 5’ exonuclease, 0.4 U – 4 U 3’ exonuclease to a reaction mixture with a buffer that contains Mg2+.
One would expect a reasonable chance of success as Goldberg discloses Fig. 3 and 5 which shows linear mRNA degradation with Xrn-1 and RNase R. Furthermore, Goldberg states that in preferred embodiment, the RNase is RNase R AND/or Xrn-1 (see paragraph 0022) and that Rnase R and XRN-1 exonucleases results in the highest purity (see paragraph 0145). Furthermore, Xiao and Dodbele discloses linear mRNA degradation with RNase R can be supplement with further modification, which can be a modification of the buffer (switching K+ ion to Li+ ion) or the addition of secondary exonucleases (see discussion of Xiao). Athapattu shows that it is necessary for the reaction buffer to contain Mg2+ for enzymatic activity of Xrn-1, which is also supported by Goldberg by the use of the NEB reaction buffer containing MgCl2.
Though the exact amount of 5’ exonuclease is not disclosed in any of the prior arts, it is obvious that through routine optimization, one would reach the amount of 0.01 U to 0.2 U 5’ exonuclease as recited in claims 1, 18, 30, and 41 of the instant application. Goldberg discloses the use of 5 U to 10 U xrn-1 in Fig. 3A, and 20 U of RNase R in Fig. 8 for digestion experiments. Xiao discloses 10 U of RNase R for digestion experiments. The instant claims also recites a wide range, from 0.01-0.2 U or 0.012 to 0.1 U which at its lowest and highest range represent almost a 10-20 times difference in the amount of 5’ exonuclease used. It is clear that one skilled in the art would understand the variable nature of enzymatic activity and the influence of factors such as enzyme concertation or buffer conditions, therefore a method of digesting linear polyribonucleotides would require optimization, leading to testing 0.01 – 0.2 U of 5’ exonuclease. One would be motivated to routinely optimize for the digestion as Dodbele discloses that there are differences in digestion efficiencies across commercial lots of exonucleases, and one should always verify that reaction conditions used in an experiment lead to efficient digestion. Dodbele also teaches that too much enzyme could lead to circular RNA degradation (see Dodbele, section titled ‘RNase R digests many, but not all linear RNAs).
Furthermore, with regards to routine optimization, please see MPEP § 2144.05 (II) where optimization with prior art conditions or through routine experimentation, such as 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. As applicant provides a varying range, applicant is silent on whether the amount or specific amount of 5’ exonuclease added provides a contribution over the prior art. 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 that will sustain a patent, even though the changes of the kind may produce better results that prior inventions”.
In view of the foregoing, claims 1-2, 6-8, 10, 14, 18, 20, 22, 30, and 41 are rejected under 35 U.S.C. 103 as being prima facie obvious before the effective filing date.
Claims 22 and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Goldberg et al. (US 2018/0169146 A1, published 6/21/2018) in view of Xiao et al. (An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3’ ends, Nucleic Acids Research, Volume 47, Issue 16, pgs. 8755-8769, published 9/19/2019), and Dodbele et al. (Best practices to ensure robust investigation of circular RNAs: pitfalls and tips, EMBO Rep. Volume 22, Issue 3, Published 2/25/2021), in further view of Goldberg et al. (US 2021/0047360 A1, published 2/18/2021).
Regarding the method of enriching a population of circular polyribonucleotides as recited in claim 1 or claim 26, Goldberg (2018) teaches the method of claim 1 except for the amount of 5’ exonuclease added, as described above. Regarding claim 26, Goldberg (2018) fully anticipates the claim as described above.
Regarding claim 1, the combined arts of Goldberg (2018), Xiao, and Dodbele teaches all limitations of claim 1, as described above.
Regarding claims 22 and 45, which depends on claim 1, Goldberg (2018) does not teach where in the resulting total population, circular polyribonucleotides makes up between 40 – 95% of the total population, or where the overall percent yield of circular polyribonucleotides is between 70 and 100%.
Regarding claims 22 and 45, Goldberg (2021) teaches a circular RNA that has been purified by a method described herein (which includes digestion with an RNase, see paragraph 0027) results in a composition comprising trace amounts of its linear form up to about 15% of its linear form. The circular RNA composition comprises between 0.1% and 10% of its linear form (see paragraph 0110), indicating that the total circular RNA population ranges from 90% to 99.9%.
It would have been obvious to one with ordinary skill in the art, to combine the teachings of Goldberg (2018) with the teachings of Xiao, Dodbele, and Goldberg (2021) to arrive at a method of producing an enriched population of circular polyribonucleotides, wherein the total population of polyribonucleotides comprises about 40% to 95% circular polyribonucleotides.
One would expect a reasonable chance of success as Goldberg (2021) shows treatment and purification with a method described herein, including treatment with RNase R and Xrn-1 (see paragraph 0027) results in a population wherein there is 0.1% to 10% linear RNA or fragments thereof and the rest of the polyribonucleotides are circular (see paragraph 0110).
One would be motivated to do so as Goldberg (2021) discloses that circularized RNA have improve stability in cells and that the methods described herein provides an optimized method for generating circularized RNA in higher yields (see paragraph 0054). Furthermore, neither claim 22 and 45 recite any additional active steps. Therefore, the recited purity of the total population is a result of the method described by Goldberg 2018 and 2021.
In view of the foregoing, claims 22 and 45 are rejected under 35 U.S.C. 103 as being prima facie obvious before the effective filing date.
Conclusion
No claims are allowed.
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/D.T.Y./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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