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 .
Application Status
The preliminary amendments filed 02 April 2024 are acknowledged and have been entered. Claims 3, 5-12 and 15 are amended. Claims 16 is newly added. Claims 1-16 are pending and being examined on the merits.
Priority
The application is a 371 PCT of CN2023/118128 filed 09/12/2023. Acknowledgment is made of applicant's claim for foreign priority based on an application filed in China on 09/13/2022. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement filed 07/02/2024 has been acknowledged.
Specification
The use of the terms Sigma, Invitrogen, Genscript, 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
Claims 7 and 10 are objected to because of the following informalities:
Claim 7 recites “at the concentration of from”. This language is awkward. It would be remedial to reword this to language such as “at a concentration of.”
Claim 10 appears to be missing “wherein” after “claim 1,”.
Appropriate correction is required.
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.
Claims 5-7, 9-12, 15 and 16 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.
Claims 5, 6 and 7 recite the limitation "the concentration" in line 2. There is insufficient antecedent basis for this limitation in the claim.
Regarding claims 9-11, the phrase "i.e." renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claims 12 and 15-16, the phrase "e.g." renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claim 16, the recitation “a reaction solution for use in a method according to claim 1” does not clearly establish whether claim 16 is intended to incorporate the process limitations of claim 1 as limitations of the reaction solution or merely identifies an intended use of the claimed composition. A reaction solution cannot itself perform all of the steps recited in method claim 1. Applicant is required to positively recite those compositional characteristics considered to distinguish the reaction solution. For examination of the prior art, however, the phrase “for use in a method according to claim 1” is interpreted as an intended use of the claimed reaction solution and does not distinguish the reaction solution from an otherwise identical prior-art reaction solution.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-14 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claim 1 broadly encompasses a DNA template encoding any “precursor RNA” that is capable of self-splicing to form circular RNA during IVT in the same reaction solution and under the same reaction conditions. The claim does not require a Group I intron, a permuted intron-exon arrangement, particular 3′ and 5′ catalytic intron fragments, particular splice sites, homology arms, spacer sequences, or another identified structural mechanism by which the precursor RNA accomplishes the claimed self-splicing. Additionally, claim 1 is not limited to the particular reaction solution disclosed in the specification, but broadly encompasses “a reaction solution” capable of permitting both in vitro transcription and self-splicing under the same reaction conditions.
The specification defines the precursor functionally as an RNA capable of self-splicing to produce circular RNA, but the structural embodiments actually disclosed employ the PIE architecture comprising Group I intron fragments and associated splice-site/exon structures. The specification explains that Group I introns are self-splicing ribozymes requiring magnesium and free guanosine and describes the PIE system as using a modified Group I intron in which the 3′ half is permuted to the 5′ half of the exon [0005–0007]. The detailed description likewise identifies the disclosed DNA template as containing a 3′ Group I intron fragment, insert sequence, and 5′ Group I intron fragment and explains that these sequences allow the precursor RNA to circularize by self-splicing [0061–0064]. The specification demonstrates possession of particular reaction solution for carrying out the disclosed Group I intron/PIE embodiments [0011-0013; 0018]. The working examples are still narrower. The precursor used experimentally was designed from the Group I intron system described by Wesselhoeft and contained the Anabaena pre-tRNA-derived permuted Group I intron together with a CVB3 IRES, GFP coding sequence, spacer sequences, and homology arms [0081]. The specification therefore provides possession of particular PIE/Group-I-intron embodiments but does not identify representative species across the broader genus of all precursor RNAs and reactions solutions capable of producing circular RNA through self-splicing during IVT, nor structural features common to that entire functional genus sufficient to show possession of its full scope.
Wesselhoeft (Wesselhoeft et al. Nature communications 9.1 (2018): 2629) further demonstrates that successful self-splicing cannot be inferred merely from designation of an RNA as a PIE precursor. Wesselhoeft initially synthesized a Group-I-intron PIE precursor but was unable to obtain splicing products and attributed the failure to the interaction of the splice sites in long intervening sequences; homology arms were consequently engineered to bring the splice sites into proximity. [pgs. 1–2, Fig. 1]. Wesselhoeft further reports that spacer design increased splicing efficiency from 46% to 87%, whereas a disruptive spacer completely abrogated splicing, and that changing the catalytic intron and internal homology altered circularization efficiency [pg. 2, Fig. 2].
Accordingly, disclosure of the particular exemplified PIE architecture does not reasonably convey possession of every RNA architecture encompassed by the functional language of claim 1.
The additional limitations in claims 2-13 directed to reaction conditions, reagent concentrations, temperature, reaction time, template removal, and purification do not limit the broadly recited “precursor RNA” of claim 2 to the structurally defined class for which the specification demonstrates possession, do not cure the written description deficiency, and are similarly rejected.
Claims 1-14 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of preparing a circular RNA, comprising providing a template DNA that comprises the following elements operably connected to each other and arranged in the following sequence: an RNA polymerase promoter, optionally a 5' homology arm, a 3' Group I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insert sequence, optionally a 3' spacer sequence, a 5' Group I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm, wherein the template DNA comprises a sequence encoding a precursor RNA, in a reaction solution, wherein the reaction solution comprises Mg2+ in a concentration of greater than 26 mM, pyrophosphatase, a RNA polymerase, an RNase inhibitor, ATP, GTP,CTP, UTP, DTT, and a monovalent cation, does not reasonably provide enablement for a method of preparing a circular RNA, comprising providing any template DNA, wherein the template DNA comprises a sequence encoding a precursor RNA, in any reaction solution. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make the invention commensurate in scope with these claims.
Nature of the invention. The invention requires two distinct biochemical processes—RNA-polymerase-mediated transcription and RNA self-splicing/circularization—to function compatibly in a single reaction environment. The specification acknowledges that conventional PIE processing uses separate IVT and circularization conditions and states that the conventional IVT reaction conditions therefore had to be optimized for one-step synthesis [0037].
Breadth of the claims. The claim is broad because it encompasses any DNA template encoding any precursor RNA capable of self-splicing into circular RNA during IVT, using unspecified IVT reagents, unspecified self-splicing architecture, and unspecified reaction conditions, provided only that IVT and self-splicing occur in the same reaction solution under the same reaction conditions. Claim 1 is not restricted to the particular Group I intron, PIE architecture, spacer arrangement, homology arms, RNA polymerase, magnesium concentration, pyrophosphatase concentration, temperature, or other conditions demonstrated in the examples. In contrast, the specification's exemplified precursor is a specific Anabaena-derived PIE construct [0081] and reaction solution comprises specific reagents [0018].
Claim 1 is not limited to the particular reaction solutions demonstrated in the specification. Rather, the recitation of “a reaction solution” is defined principally by the desired result, i.e., that the solution permits synthesis of the precursor RNA by both in vitro transcription and self-splicing under the same reaction conditions. Claim 1 therefore encompasses any reaction solution capable of accomplishing these functions, without requiring a particular reagent or a particular amount of that reagent.
Guidance from the specification. The specification provides substantial guidance for the disclosed Group-I-intron PIE implementation and its reaction optimization but does not provide a general structural rule permitting the skilled artisan to identify all other precursor RNAs encompassed by claim 1.
The specification does not establish that the reaction solutions across this full scope are operative. The specification recognizes that conventional IVT and circularization employ different reaction conditions and demonstrates that development of the claimed one -step process required optimization of the reaction environment [0037].
Working examples. Working examples are provided, but they concern the particular PIE precursor and systematic optimization of Mg²⁺, temperature, pyrophosphatase, and polymerase. Applicants themselves tested final Mg²⁺ concentrations of 26, 36, 46, 56, 66, and 86 mM and found only 7.5% circularization at 26 mM compared with 41.8% at 36 mM, while further increases could reduce RNA production [0087–0089]. A subsequent factorial experiment varied temperature, Mg²⁺, polymerase, and pyrophosphatase and produced substantially different circularization efficiencies [Table 2; 0092–0095]. Thus, the examples themselves demonstrate substantial dependence upon multiple interacting variables.
State of the prior art and predictability. Wesselhoeft teaches that a conventional PIE precursor initially failed to yield splicing products, notwithstanding use of a known Group I catalytic intron, and that bringing the splice sites together using engineered homology arms was necessary to obtain efficient circularization [pp. 1–2, Fig. 1]. Wesselhoeft further demonstrates that rationally designed spacers increased splicing efficiency whereas a disruptive spacer completely abolished splicing [p. 2, Fig. 2]. Circularization also depended upon insert size, with longer RNAs circularizing less efficiently, and long circular RNAs were more prone to Mg²⁺-associated nicking. These teachings establish that successful circularization depends upon RNA structure, intron identity, spacer/homology architecture, insert length, and reaction chemistry and is therefore not reasonably predictable across the full functional scope of claim 1.
Level of skill. The level of skill was relatively high, as evidenced by the sophisticated IVT and ribozyme engineering known in the art. This factor weighs toward enablement but does not eliminate the need to discover which uncharacterized precursor architectures and reaction conditions successfully perform the claimed dual reaction.
Quantity of experimentation. To practice the full scope, the skilled artisan would be required to identify alternative precursor architectures, construct them, determine whether they self-splice during IVT, and, for unsuccessful constructs, vary intron architecture, splice-site proximity, RNA folding, spacer/homology sequences, Mg²⁺, temperature, polymerase, and other reaction conditions until successful one-step circularization is obtained. Such experimentation is not merely verification of embodiments taught by the specification but constitutes screening and optimization to discover which members of the broad functional genus work.
The evidence as a whole therefore establishes that the scope of enablement is not commensurate with the scope of claim 1.
Although the dependent claims, claims 2-14, further limit the precursor RNA or individual reaction parameters, such limitations do not necessarily cure the separate enablement deficiency because such limitations for not restrict the broadly claimed precursor RNA and reaction condition to a structurally defined or specific genus demonstrates to have been possessed and enabled by the specification and are similarly rejected.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 16 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 16 is directed to a reaction solution and depends from the method of claim 1. The reaction solution does not comprise the method steps of preparing a circular RNA. Therefore claim 16 fails to include all the limitations of the claim upon which it depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 1, 2 and 9–11 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Wei (WO 2023/115732, filed 3/22/2022).
Regarding claim 1, Wei teaches a method of producing a circular RNA from a DNA construct encoding a linear RNA precursor, wherein the precursor comprises, from the 5′ end to the 3′ end, a 3′ catalytic Group I intron fragment, a 3′ exon sequence, an effector RNA sequence, a 5′ exon sequence, and a 5′ catalytic Group I intron fragment [claim 1]. Wei further teaches contacting the DNA construct, in a single-pot reaction, with a reagent composition comprising an RNA polymerase and ATP, UTP, GTP, and CTP under conditions that allow both transcription of the DNA construct into the linear RNA precursor and circularization of the linear RNA precursor through activation and self-splicing of the Group I intron fragments [claim 1]. Wei expressly describes the disclosed process as a method for producing circular RNA using a single-pot in vitro transcription reaction, in contrast to known methods requiring separate IVT, DNase I treatment, and GTP/divalent-metal circularization steps [0025–0026]. Wei defines “single-pot reaction” as multiple reaction or synthesis steps taking place within the same reaction vessel without subsequent separation and/or purification of intermediary compounds [0031]. Wei further explains that during the single-pot process, the NTP mixture used for IVT is sufficient to permit activation of self-splicing of the Group I intron fragments, and the divalent metal ion already present in the reaction composition may be Mg²⁺, such that a separate GTP/divalent-metal treatment is unnecessary [0048–0052]. Accordingly, Wei teaches providing a DNA template encoding a precursor RNA in a reaction solution, synthesizing the precursor RNA by in vitro transcription, allowing the precursor RNA to self-splice to produce circular RNA, and carrying out the IVT and self-splicing in the same reaction solution under the same reaction conditions.
Regarding claim 2, Wei teaches the single-pot reaction as occurring without subsequent separation or purification of intermediary compounds [0031] and further teaches embodiments in which the process does not comprise isolating or purifying the linear RNA precursor prior to circularization [0048, p. 10].
Regarding claim 9, Wei teaches that the DNA construct may be contacted with the single-pot reagent composition at temperatures including 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, and temperatures extending through and beyond 55°C [0059]. Wei further expressly claims contacting the DNA construct with the reagent composition at about 37°C [claim 9]. Therefore, Wei teaches carrying out the IVT and circularization at a temperature within the claimed range of 37°C to 55°C.
Regarding claim 10, Wei teaches performing the single-pot reaction at temperatures greater than 37°C, including 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, and 50°C [0059]. These expressly disclosed temperatures fall within the claimed range greater than 37°C and within the recited preferred range of 39°C to 50°C.
Regarding claim 11, Wei teaches conducting the single-pot reaction for numerous reaction times including 1 hour, 2 hours, 3 hours, 4 hours, and longer, and teaches reaction periods extending from minutes up to approximately 24 hours [0060]. Wei's claim 10 additionally recites contacting the DNA construct with the reagent composition for at least about 20 minutes and optionally up to about 24 hours [claim 10, p. 37].
Claims 15 and 16 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Alayyoubi et al. (US 2024/0043892 A1, provisionally filed 7/1/2022).
Regarding claims 15 and 16, Alayyoubi teaches an aqueous reaction solution for in vitro transcription comprising magnesium ions, pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, a reducing agent, and a monovalent cation. Specifically, Alayyoubi teaches preparing the IVT reaction using molecular-biology-grade water and a reaction mixture comprising magnesium acetate, ATP, CTP, GTP, UTP, T7 RNA polymerase, RNase inhibitor, inorganic pyrophosphatase, and DTT [Example 1; Tables 1–2]. Alayyoubi teaches a 10 g/L IVT condition comprising 50 mM magnesium acetate and 10 mM each ATP, CTP, GTP, and UTP, and a 15 g/L condition comprising 65 mM magnesium acetate and 15 mM each ATP, CTP, GTP, and UTP [Table 2]. Magnesium acetate is expressly identified by Alayyoubi as a suitable source of Mg²⁺ for IVT [0085–0086]. Because magnesium acetate contains one magnesium ion per formula unit, the disclosed 50 mM and 65 mM magnesium acetate concentrations provide nominal magnesium-ion concentrations of 50 mM and 65 mM, respectively, which are greater than 26 mM and fall within the recited range of 38–66 mM. Alayyoubi further teaches an actual IVT reaction comprising 50 mM MgOAc, 10 mM each NTP, 0.025 μg/μL T7 polymerase, 40 mM Tris at pH 7.6, 10 mM DTT, 0.25 μg/μL RNase inhibitor, and 0.002 U/μL inorganic pyrophosphatase [0200; Table 4]. The disclosed 0.002 U/μL pyrophosphatase corresponds to 2 U/mL, which falls within the recited exemplary concentration of 1–4 U/mL. Alayyoubi further teaches introducing linear DNA containing NaCl into the foregoing IVT reaction, including L.DNA spiked with 200 mM NaCl [0200; Table 4]. NaCl dissociates in aqueous solution to provide Na⁺, which is a monovalent cation and therefore satisfies the claimed requirement for a monovalent cation. Thus, Alayyoubi teaches an aqueous reaction solution comprising Mg²⁺ at a concentration greater than 26 mM, including concentrations within 38–66 mM; pyrophosphatase, including approximately 2 U/mL; RNA polymerase; RNase inhibitor; ATP, GTP, CTP and UTP; DTT as a reducing agent; and a monovalent cation.
The recitation that the reaction solution is “for use in a one-step circular RNA synthesis” merely states an intended use of the claimed reaction solution and does not impart a structural or compositional distinction over the otherwise identical reaction solution disclosed by Alayyoubi.
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.
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.
Claims 1-4 and 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over Wei (WO 2023/115732, filed 3/22/2022) in view of Wesselhoeft (Wesselhoeft et al., Nature communications 9.1 (2018): 2629).
Regarding claim 1, Wei teaches a method of producing a circular RNA from a DNA construct encoding a linear RNA precursor, wherein the precursor comprises, from the 5′ end to the 3′ end, a 3′ catalytic Group I intron fragment, a 3′ exon sequence, an effector RNA sequence, a 5′ exon sequence, and a 5′ catalytic Group I intron fragment [claim 1]. Wei further teaches contacting the DNA construct, in a single-pot reaction, with a reagent composition comprising an RNA polymerase and ATP, UTP, GTP, and CTP under conditions that allow both transcription of the DNA construct into the linear RNA precursor and circularization of the linear RNA precursor through activation and self-splicing of the Group I intron fragments [claim 1]. Wei expressly describes the disclosed process as a method for producing circular RNA using a single-pot in vitro transcription reaction, in contrast to known methods requiring separate IVT, DNase I treatment, and GTP/divalent-metal circularization steps [0025–0026]. Wei defines “single-pot reaction” as multiple reaction or synthesis steps taking place within the same reaction vessel without subsequent separation and/or purification of intermediary compounds [0031]. Wei further explains that during the single-pot process, the NTP mixture used for IVT is sufficient to permit activation of self-splicing of the Group I intron fragments, and the divalent metal ion already present in the reaction composition may be Mg²⁺, such that a separate GTP/divalent-metal treatment is unnecessary [0048–0052]. Accordingly, Wei teaches providing a DNA template encoding a precursor RNA in a reaction solution, synthesizing the precursor RNA by in vitro transcription, allowing the precursor RNA to self-splice to produce circular RNA, and carrying out the IVT and self-splicing in the same reaction solution under the same reaction conditions.
Regarding claim 2, Wei teaches the single-pot reaction as occurring without subsequent separation or purification of intermediary compounds [0031] and further teaches embodiments in which the process does not comprise isolating or purifying the linear RNA precursor prior to circularization [0048, p. 10].
Regarding claim 9, Wei teaches that the DNA construct may be contacted with the single-pot reagent composition at temperatures including 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, and temperatures extending through and beyond 55°C [0059]. Wei further expressly claims contacting the DNA construct with the reagent composition at about 37°C [claim 9]. Therefore, Wei teaches carrying out the IVT and circularization at a temperature within the claimed range of 37°C to 55°C.
Regarding claim 10, Wei teaches performing the single-pot reaction at temperatures greater than 37°C, including 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, and 50°C [¶0059, pp. 12–13]. These expressly disclosed temperatures fall within the claimed range greater than 37°C and within the recited preferred range of 39°C to 50°C.
Regarding claim 11, Wei teaches conducting the single-pot reaction for numerous reaction times including 1 hour, 2 hours, 3 hours, 4 hours, and longer, and teaches reaction periods extending from minutes up to approximately 24 hours [0060]. Wei's claim 10 additionally recites contacting the DNA construct with the reagent composition for at least about 20 minutes and optionally up to about 24 hours [claim 10, p. 37].
Regarding claim 3-4, Wei does not teach where the DNA template comprises the following elements operably connected to each other and arranged in the following sequence: an RNA polymerase promoter, optionally a 5' homology arm, a 3' Group I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insert sequence, optionally a 3' spacer sequence, a 5' Group I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm; or wherein the insert sequence comprises a protein coding sequence, and wherein the insert sequence comprises an IRES sequence operably connected to the protein-coding sequence.
Wei does teach the Group-I-intron PIE arrangement and expressly claims 5′ and 3′ homology arms flanking the catalytic intron fragments and hybridizing to one another [claims 16–20; Fig. 1].
Wesselhoeft teaches the detailed PIE precursor comprising 3′ and 5′ halves of a Group I catalytic intron, exon fragments, homology arms, and engineered spacer sequences. Wesselhoeft explains that homology arms were introduced to bring the 5′ and 3′ splice sites into proximity [p. 2, Fig. 1]. Wesselhoeft further teaches engineered spacer sequences designed to separate intron and IRES structures and promote formation of a splicing bubble, together with 5′ and 3′ homology arms [p. 2-5, Fig. 3]. Wesselhoeft expressly teaches an EMCV IRES positioned with protein-coding regions and successfully circularized constructs encoding Gaussia luciferase, Firefly luciferase, eGFP, human erythropoietin, and Cas9 [p. 3, Fig. 3]. Wesselhoeft teaches that the use of untranslated regions increases mRNA stability, and spacer sequences with homology arms improve circularization efficiency [pg. 2].
It would have been obvious to employ Wesselhoeft's known homology arms, spacers, IRES, and protein-coding inserts in Wei's single-pot Group-I-intron process because Wesselhoeft expressly teaches these structural features as improving PIE folding/splicing and permitting efficient circularization of diverse protein-coding inserts.
Claims 5-8 are rejected under 35 U.S.C. 103 as being unpatentable over Wei (WO 2023/115732, filed 3/22/2022) in view of Alayyoubi et al. (US 2024/0043892 A1, provisionally filed 7/1/2022).
The teachings of Wei are discussed above as applied to claim 1 and similarly apply to claims 5-8.
Wei do not teach wherein the reaction solution comprises Mg2+ at the concentration of 3 8-66 mM, optionally 1-4 U /ml pyrophosphatase, an RNA polymerase, an RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and a monovalent cation (Na+ or K+).
Alayyoubi teaches an aqueous reaction solution for in vitro transcription comprising magnesium ions, pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, a reducing agent, and a monovalent cation. Specifically, Alayyoubi teaches preparing the IVT reaction using molecular-biology-grade water and a reaction mixture comprising magnesium acetate, ATP, CTP, GTP, UTP, T7 RNA polymerase, RNase inhibitor, inorganic pyrophosphatase, and DTT [Example 1; Tables 1–2]. Alayyoubi teaches a 10 g/L IVT condition comprising 50 mM magnesium acetate and 10 mM each ATP, CTP, GTP, and UTP, and a 15 g/L condition comprising 65 mM magnesium acetate and 15 mM each ATP, CTP, GTP, and UTP [Table 2]. Magnesium acetate is expressly identified by Alayyoubi as a suitable source of Mg²⁺ for IVT [0085–0086]. Because magnesium acetate contains one magnesium ion per formula unit, the disclosed 50 mM and 65 mM magnesium acetate concentrations provide nominal magnesium-ion concentrations of 50 mM and 65 mM, respectively, which are greater than 26 mM and fall within the recited range of 38–66 mM. Alayyoubi further teaches an actual IVT reaction comprising 50 mM MgOAc, 10 mM each NTP, 0.025 μg/μL T7 polymerase, 40 mM Tris at pH 7.6, 10 mM DTT, 0.25 μg/μL RNase inhibitor, and 0.002 U/μL inorganic pyrophosphatase [0200; Table 4]. The disclosed 0.002 U/μL pyrophosphatase corresponds to 2 U/mL, which falls within the recited exemplary concentration of 1–4 U/mL. Alayyoubi further teaches introducing linear DNA containing NaCl into the foregoing IVT reaction, including L.DNA spiked with 200 mM NaCl [0200; Table 4]. NaCl dissociates in aqueous solution to provide Na⁺, which is a monovalent cation and therefore satisfies the claimed requirement for a monovalent cation. Thus, Alayyoubi teaches an aqueous reaction solution comprising Mg²⁺ at a concentration greater than 26 mM, including concentrations within 38–66 mM; pyrophosphatase, including approximately 2 U/mL; RNA polymerase; RNase inhibitor; ATP, GTP, CTP and UTP; DTT as a reducing agent; and a monovalent cation.
It would have been obvious to employ Alayyoubi's known high-magnesium IVT, pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cation conditions, in Wei's single-pot IVT/circularization system because Wei requires an IVT-compatible reaction environment and Alayyoubi teaches specific reagents and concentrations as workable IVT conditions in the presence of elevated NTP concentrations. The modification amounts to employing known IVT reaction chemistry for its known purpose—supporting RNA-polymerase-mediated transcription—within Wei's expressly disclosed single-pot IVT reaction.
Claims 12–14 are rejected under 35 U.S.C. 103 as being unpatentable over Wei (WO 2023/115732, filed 3/22/2022) in view of Eber (US 2018/0201967 A1).
The teachings of Wei are discussed above as applied to claim 1 and similarly apply to claims 12-14.
Wei do not teach wherein the method further comprises a step of removing the DNA template after synthesis of the precursor RNA, optionally wherein the DNA template is removed by adding a DNase I, e.g., for 30 min at 37 °C; wherein the method further comprises a step of purifying the circular RNA after the step of removing the DNA template; or wherein the purification step is selected from a precipitation step, a tangential flow filtration step and a chromatographic step, and a combination thereof.
Eber teaches that after a three-hour IVT reaction, DNase I is added and incubated at 37°C for 30 minutes, thereby removing/degrading the DNA template, after which RNA is precipitated with LiCl [0246]. Eber also teaches chromatographic RNA purification, including HPLC, followed by precipitation [; claims 8-9; 0026].
It would have been obvious to apply Eber's conventional post-IVT DNase treatment and RNA purification to Wei's single-pot circRNA product because, after completion of Wei's IVT/circularization reaction, residual template DNA and reaction contaminants remain and Eber teaches DNase treatment followed by RNA purification for precisely the conventional purposes of removing template DNA and recovering purified RNA.
Regarding claim 14, the claimed alternatives are satisfied by disclosure of any one alternative. Eber teaches precipitation and chromatography. Wesselhoeft independently teaches purification of circular RNA from splicing reactions by gel extraction and size-exclusion HPLC, followed by ammonium-acetate precipitation [Methods].
Accordingly, claims 12–14 would have been obvious over Wei in view of Eber, with Wesselhoeft providing additional evidence that chromatographic purification was specifically known for PIE-derived circular RNA.
Conclusion
No claims allowed.
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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
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