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 .
Claim Status
Claim 6 is amended.
Claims 1-13 are examined on the merits.
Priority
The applicant’s application is a U.S. National Stage application of PCT International Patent Application Serial No. PCT/KR2023/019046, filed November 24, 2023, which itself claims the benefit of Korean Patent Application Serial No. KR10-2022-0187737, filed December 28, 2022, is acknowledged.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings. See Figs. 2A to 2P.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. See page 7 at lines 24 and 27.
Claim Objections
Claim 1 is objected to because of the following informalities: reciting an abbreviation without spelling out what the abbreviation is in the first occurrence in the claim set (GOI).
Claims 2-13 depend from claim 1 and are objected to for the same reason applied to claim 1.
Claim 5 is objected to because of the following informalities: reciting an abbreviation without spelling out what the abbreviation is in the first occurrence in the claim set (IRES).
Claim 9 recites: The method for preparing circular RNA of claim 8, further comprising: treating RppH in vitro after the 2 is performed. "the 2" is intended to refer to step (2) of claim 8.
It would be preferable to amend claim 9 to recite, "The method for preparing circular RNA of claim 8, further comprising: treatment with RppH in vitro after step (2) is performed."
Claim 10 recites: The method for preparing circular RNA of claim 8, further comprising: heating immediately before the 3, wherein the heating is performed by heating at 80◦C for 3 minutes and cooling to 26◦C by 6◦C per minute. “The 3” is intended to refer to step (3) of claim 8.
It would be preferable to amend claim 10 to recite, "The method for preparing circular RNA of claim 8, further comprising: heating immediately before step (3), wherein the heating is performed ◦C for 3 minutes and cooling to 26◦C by 6◦C per minute."
Claim 11 recites: The method for preparing circular RNA of claim 8, wherein the 3 is performed at 25◦C for 1 h or more. “The 3” is intended to refer to step (3) of claim 8.
It would be preferable to amend claim 11 to recite, “The method for preparing circular RNA of claim 8, wherein step (3) is performed at 25◦C for 1 h or more.
Claim 12 recites: The method for preparing circular RNA of claim 8, further comprising: (4) treating RNase R in vitro after the 3. “The 3” is intended to refer to step (3) of claim 8.
It would be preferable to amend claim 12 to recite, “The method for preparing circular RNA of claim 8, further comprising: (4) treatment with RNase R in vitro after step (3).
Claim 13 recites: The method for preparing circular RNA of claim 12, further comprising: treating poly (A) polymerase in vitro immediately before the 4. “The 4” is intended to refer to step (4) of claim 12.
It would be preferable to amend claim 13 to recite, “The method for preparing circular RNA of claim 12, further comprising: treatment with poly (A) polymerase in vitro immediately before step (4).
Appropriate correction is required.
Claims 2-3 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The closest prior are Chen et al. (NAR, 2020) discloses that too many base pairs in the 5’-phosphate side (5’DB SS) are unfavorable to suppress the intermolecular ligation by Rnl2 (e.g., paragraph 2nd, column right, page 5; Fig. 4).
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Claims 1, 4-8, 11-12 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (“Chen”, Nucleic Acids Research, 2020) in view of Anderson et al. (“Anderson”, WO 2019/236673 A1).
Regarding claim 1, Chen teaches functional RNAs (siRNA, miRNA, ribozyme, etc.) are dominantly converted by Rnl2 to the rings without significant limitations in sizes and sequences (e.g., abstract). Chen teaches cyclic intermediate structure as strategy to circularize linear-RNA (L-RNA) to circular-RNA (C-RNA) by Rnl2 (e.g., Scheme 1B [see below]).
PNG
media_image1.png
200
400
media_image1.png
Greyscale
Chen teaches that the base pairs in the 3’-OH side should be two or larger (e.g., paragraph 3rd, column right, page 5; Figs. 1-3). On the other hand, the base pairs in the 5’-phosphate side should be kept 0 or 1, since further increase in the number deteriorates the selectivity. Too many base pairs (e.g. ≥4 bp) at 3’-OH side may also increase polymeric by-products (e.g., paragraph 1st, column left; Supplementary Fig. S2B and C) (It reads on that 3’DB SS should be 2 nt or larger).
Regarding claims 8 and 11, Chen teaches circularization of L-RNA by T4 Rnl2 at 25◦C for 2 h (unless noted otherwise) and terminated by heating the mixture at 75◦C for 10 min (e.g., paragraph 2nd, column right, page 3).
Chen does not teach that the length of the GOI region of 500 to 5500 nt. Chen does not teach that that the GOI includes an IRES. Chen does not teach that an expression vector capable of expressing the circular RNA precursor. Chen does not teach that an expression vector comprising a promoter linked to gene encoding the circular RNA precursor, as required by the instant claims. Chen does not teach a method for preparing circular RNA comprising: (1) inducing in vitro transcription of the expression vector of claim 6 under a condition of GTP : GMP = 1 : 5 to 10; (2) treating DNase I in vitro after the transcription induction is completed. Chen does not teach the method for preparing circular RNA of claim 8, further comprising: (4) treating RNase R in vitro after the 3. However, this is cured by Anderson.
Regarding claims 1, 4-5, Anderson teaches a method of making circular RNA with enhanced translation efficiency, said method comprising incorporating artificial nucleosides into a precursor RNA during transcription of a vector encoding the precursor RNA and circularizing the precursor RNA to form the circular RNA (e.g., paragraph 0041). Anderson teaches precursor RNA containing an EMCV IRES and a variable insert including Gaussia luciferase (GLuc), human erythropoietin (hEpo), EGFP, Firefly luciferase (FLuc ), or Cas9 coding regions after circularization and recircularization (R-) (e.g., paragraph 0059; Fig. 2B) (It reads on [GOI], large protein-coding inserts can be incorporated into the precursor circular RNA [between the 5’ and 3’ ends of the precursor RNA], the size of SpCas9 open reading frame is ~4,200 nucleotides).
Regarding claim 6, Anderson teaches the precursor RNA is a circular RNA produced by in vitro transcription of a vector (e.g., paragraph 0033).
Regarding claim 7, Anderson teaches the vectors provided herein comprise an RNA polymerase promoter upstream of the region that encodes the precursor RNA (e.g., upstream of the 5' homology arm). In some embodiments, the promoter can be recognized by the T7 phage RNA
polymerase enzyme (e.g., paragraph 0045).
It would have been obvious to one of ordinary skill in the art at the time of the invention to employ the vector for making circular RNA that encodes a protein of interest taught by Anderson to express the dumbbell structure of Chen, because, both Chen and Anderson recognized that relevance of functional circular RNAs and that circularization reaction is not limited by RNA size or sequence. Anderson teaches a precursor RNA comprising an RNA promoter upstream of an IRES-gene encoding protein of interest that can be expressed by a vector. One of ordinary skill in the art would have had a reasonable expectation that the vector expressing the modified precursor having the claimed dumbbell structure, with an RNA promoter upstream of an IRES-gene encoding protein of interest having a length within the range of 500 to 5000 nucleotides as taught by Anderson, would have produce predictable results.
Regarding claim 8, Anderson teaches a method of generating precursor RNA by performing in vitro transcription using a vector provided as a template (e.g., paragraph 00142). Anderson teaches 1) the use of a nucleotide, nucleoside, or a chemically modified nucleotide or nucleoside in the in vitro transcription reactions described herein is at an excess concentration relative to the analogous nucleotide triphosphate (e.g., paragraph 00143). Anderson teaches that the nucleotide used in excess is guanosine monophosphate (GMP). In other embodiments, the nucleotide used in excess is GDP, ADP, CDP, UDP, AMP, CMP, UMP, guanosine, adenosine, cytidine, uridine, or any chemically modified nucleotide or nucleoside. In some embodiments, the excess is about a 10-fold excess (e.g., paragraph 00144). Anderson teaches 2) after in vitro transcription, reactions were treated with DNase I for 20 minutes (e.g., paragraph 00192). 3) Anderson teaches an alternative method of circRNA preparation using T4 RNA ligase I and splint oligonucleotides designed to bring the ends of the precursor RNA into proximity for ligation (e.g., paragraph 00219).
It would have been obvious to one of ordinary skill in the art at the time of the invention to combine the in vitro transcription method of Anderson with the enzymatic circularization method of Chen. A person of ordinary skill in the art would have been motivated to perform an in vitro transcription using the expression vector in presence of an excess ratio of GTP:GMP of 1:10 as taught by Anderson in order to ensure efficient 5’ monophosphorylation of the transcript which is required as substrate for Rn12 circularization as taught by Chen. A person of ordinary skill would have found routine to incorporate DNase I treatment step following transcription to eliminate the expression vector.
Regarding claim 12, Anderson teaches purity of circRNA preparations is another factor essential for maximizing protein production from circRNA and for avoiding innate cellular immune responses. Purification was followed with RNase R treatment to eliminate the majority of degraded RNA (e.g., paragraph 00175) (It reads on treatment of circular RNA with RNase R).
It would have been obvious to one of ordinary skill in the art at the time of the invention to include an RNase R treatment step following the Rnl2 ligation step (3). A person of ordinary skill in the art would have been motivated to use RNase R to selectively degrade non-circularized linear precursor, therefore purifying the desired circular RNA product.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (“Chen”, Nucleic Acids Research, 2020) and Anderson et al. (“Anderson”, WO 2019/236673 A1) as applied to claims 1, 4-8, 11-12 above, and further in view of Carmona, M. (“Carmona”, Doctoral dissertation, Harvard University, 2019).
Chen and Anderson do not teach treatment with RppH in vitro, as required by the instant claim. However, this is cured by Carmona.
Carmona teaches proof-of-concept in vivo studies using circRNA encoding erythropoietin (EPO) showed significantly longer protein expression and elevated reticulocyte levels compared to standard, modified linear mRNA (e.g., abstract). Carmona teaches that all forms of enzymatic ligation (ssRNA and splint-mediated) require a 5’ monophosphate end to undergo ligation, which means two phosphates must be removed from the linear, triphosphate RNA that is generated by in vitro transcription (e.g., paragraph 3rd, page 19). Carmona teaches that enzymes derived from E. coli, RNA Pyrophosphohydrolase (RppH) and Apyrase, were said to be able to generate a 5’ monophosphate end in a single reaction with great efficiency. RppH was by far the superior method for obtaining 5’ monophosphate ends. Further testing using XRN-1, a 5’ à 3’ exonuclease that specifically targets 5’ monophosphate ends, showed that RppH and Apyrase were capable of cleaving the g and b phosphates with almost 100% efficiency. This optimization was one in a series of successful attempts to enhance the ligation efficiency (e.g., paragraph 2nd, page 20; Figs. 5C-D).
It would have been obvious to one of ordinary skill in the art at the time of the invention to combine the in vitro transcription method of Anderson with the enzymatic treatment with RppH taught by Carmona to obtain a circular RNA. A person of ordinary skill in the art would have been motivated to include a treatment of the transcript/DNase I generated by steps (1) and (2) to generate 5’ monophosphate to enhance the circularization efficiency as taught by Carmona.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (“Chen”, Nucleic Acids Research, 2020) and Anderson et al. (“Anderson”, WO 2019/236673 A1) as applied to claims 1, 4-8, 11-12 above, and further in view of Huang et al. (“Huang”, BioTechniques, June 1996).
Anderson teaches that after DNase treatment; unmodified linear mRNA was column purified using a MEGAclear Transcription Clean-up kit. RNA was then heated to 70°C for 5 minutes and immediately placed on ice for 3 minutes (e.g., paragraph 00192).
Chen and Anderson do not teach heating is performed by heating at 80◦C for 3 minutes and cooling to 26◦C by 6◦C per minute, as required by the instant claim. However, this is cured by Huang.
Huang teaches that although inactivation of DNase I at either 75°C or 85°C preserved almost all message, 75°C was slightly better and is therefore recommended as the optimal temperature. Essentially, no effect on the mRNA during at least 20 min of thermal inactivation and up to 5 U/mg RNA of DNase I (e.g., paragraph 1st, right column, page 6; Figs. 1-2)
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the method of Anderson of heating at 70◦C for 5 minutes and immediately place on ice for 3 minutes with the teachings of Huang a thermal inactivation of DNase I at temperatures of approximately 75◦C or 85◦C, because Anderson provides an expressly teaching of heating RNA after DNase treatment, while Huang teaches that elevated temperature in the 75◦-85◦C range can be used for thermal inactivation of DNase I without substantially compromising RNA. Thus, Huang would have provided a reason to increase Anderson’s 70◦C heating temperature to a temperature in the range of 75◦-85◦C range, including 80◦C, particularly where the goal is to ensure effective thermal inactivation of residual DNase I before proceeding to the ligation step. Furthermore, modifying Anderson’s 5-minute heating period to 3 minutes would have been an optimization of the duration of a known thermal treatment. The selection of a particular temperature/time combination within these teachings would have been within the routine skill of one of ordinary skill in the art. With respect to the cooling condition, Anderson teaches that RNA is immediately placed on ice after heating, demonstrating the desirability of rapidly reducing the temperature of the RNA following thermal treatment. Therefore, it would have been obvious to control the cooling temperature of the RNA preparation following the 80◦C treatment to return the RNA to a temperature suitable for the subsequent Rnl2 ligation reaction, including cooling to 26◦C. Selecting a cooling rate of 6◦C per minute represents optimization of a parameter to achieve the desired reaction temperature and would have been a routine matter of process optimization.
Accordingly, one of ordinary skill in the art would have had a reasonable expectation of success in combining the teachings of Chen, Anderson and Huang to arrive at the method of claim 10. The claimed heating temperature, duration, and cooling conditions represent optimization of a known RNA-processing parameters rather than a patentably distinct process.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (“Chen”, Nucleic Acids Research, 2020) and Anderson et al. (“Anderson”, WO 2019/236673 A1) as applied to claims 1, 4-8, 11-12 above, and further in view of De Boer et al. (“De Boer”, WO 2022/051629 A1).
Chen and Anderson do not teach treatment with poly (A) polymerase in vitro, as required by the instant claims. However, this is cured by De Boer.
De Boer teaches compositions, pharmaceutical preparations, and uses of polyribonucleotides encoding one or more immunogenic polypeptides. In particular, this disclosure features circular polyribonucleotide encoding one or more immunogenic polypeptide (e.g., abstract). De Boer teaches that circular polyribonucleotide is purified by utilizing a structural feature of the circular polyribonucleotide to separate it from a linear RNA or an impurity. In some embodiments, the circular polyribonucleotide is purified by utilizing a structural feature (e.g., a lack of free ends). For example, circular RNA is enriched from a preparation including a mixed pool of circular RNA and linear RNA counterpart containing the same nucleotide sequences using polyadenylation of the linear RNA counterpart or fragments thereof. This addition biotinylated N6-ATP analog into the 3' polyadenine tail enables a pulldown of the linear RNA and fragments thereof in a system such as a biotin-streptavidin binding system. In contrast, circularized RNA does not have a 3' end, and therefore is not polyadenylated by the poly(A) polymerase, does not have a polyadenylated tail for conjugation, and is not captured in the pulldown. Therefore, the circular RNA is enriched in the preparation after the pulldown (e.g., lines [16]-[30], page 92).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the circular RNA preparation method of Chen and Anderson by adding a poly(A) polymerase treatment step immediately prior to the RNase R treatment of step (4) as taught by De Boer. A person of ordinary skill in the art would have been motivated to treat the transcripts mixture of step (1) with poly(A) polymerase that selectively adenylates 3’ ends of any unreacted linear precursor or linear RNA fragments without altering the circularized RNA. Adding poly(A) tails directly creates accessible polyadenylated linear single-stranded RNA, therefore, the circular RNA is enriched in the preparation as taught by De Boer.
Allowable Subject Matter
Claims 2 and 3 are free of the prior art. The closest prior Chen et al. (NAR, 2020) teaches the effects of the numbers of terminal base pairs on the circularization efficiency by T4 Rnl2. (A) Proposed structures of L-miR-21c(3,4), L-miR-21c(2,3), L-miR-21c(1,2) and L-miR-21c(22,1). The numbers of base pairs in the 3′-OH and 5′-phosphate sides of ligation site are (5,0), (4,1), (3,2) and (2,3), respectively (e.g., Fig. 4, page 6). Chen teaches L-miR-21c(22,1) (3 bp in the 5’-phosphate side of the ligation site) was mostly converted to the polymers, and only minimal amount of C-miR-21c was obtained. On the other hand, the selectivity for the circularization increased to around 60% for LmiR-21c(1,2) (2 bp in the 5’-phosphate side; Fig. 4, lane 6). With the precursors having 0 or 1 bp in the 5-phosphate side (L-miR-21c(3,4) and L-miR-21c(2,3)), the formation of polymeric byproducts was minimized (e.g., paragraph 2nd, column right, page 5; Fig. 4). Chen teaches that an excessive number of base pairs on the 5’ phosphate side is unfavorable for suppressing the unwanted intermolecular ligation by Rnl2, Chen’s disclosed data directly teaches away from and is contrary to the 3 to 15 nucleotides length of the 5’DB SS recited in the instant claims.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JULIO GOMEZ RODRIGUEZ whose telephone number is (571)270-0991. The examiner can normally be reached Monday - Friday 8:00 am - 5:00 pm.
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, Jennifer Dunston can be reached at 5712722916. 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.
/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
