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 .
Response to Amendment
Receipt of Arguments/Remarks filed on 09/26/2025 is acknowledged. Claims 1-10,13 and 22-30 stand cancelled. No claims were amended. Claims 11,12 and 14-21 are pending.
Applicant elected SEQ ID NO: 1 with traverse in the reply filed on 08/29/2024 and the species election requirement was maintained. Upon further consideration, the species election requirement has been withdrawn.
Response to Arguments
Applicant’s arguments, see pages 2-3, filed 09/26/2025, with respect to the rejection(s) of claim(s) 11,12 and 14-18 under 35 U.S.C. 103 as obvious over US 20040241651, Fan et al., and Soboleski et al., claims 19-20 as obvious over ‘651, Fan and Soboleski further in view of Yang et al., and claim 21 as obvious over ‘651, Fan, Soboleski and Yang, and further in view of US 20170298347 have been fully considered and are persuasive as the ‘651 publication does not suggest the disclosed oliognuceotides/oligomers can be used to construct an RNA construct, and has not provided a case of obviousness regarding using a primer/probe sequence suitable for SNP/methylation detection as a translation initiation element in a circular RNA construct. Therefore, the 103 rejections have been withdrawn.
However, the examiner is issuing another non-final office action in view of a new case of obviousness for the instant claims. See the new 103 rejections below.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
However, there is no English translation of the non-English language foreign priority application.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Applicant only enjoys priority to PCT/CN2020/077026 filed on 02/27/20.
Claims 11,12 and 14-18 are rejected under 35 U.S.C. 103 as being unpatentable over Fan et al. (Preprint from “bioRxiv” online journal, 18 Nov 2018), cited on an IDS and in previous office action, in view of the English translation of Guo et al. (CN109022478, Published 18 Dec 2018) cited on an IDS dated 09/26/2025, and Noderer et al. (Mol. Syst Biol. 28 Aug 2014; 10(8):748).
Regarding claims 11,12 and 14, Fan et al. teach circular RNAs are stable due to resistance to the degradation by exonucleases (Intro, page 2) and that some circRNAs have been shown to be translated through IRES driven cap-independent translation, and teach the screening of random 10-nt library using a cell-based reporter system and identified a large number of AU-rich motifs capable of initiating circRNA translation, and that these IRES-like elements are significantly enriched in circRNAs vs linear mRNA and are sufficient to drive robust translation of circRNAs containing solely the coding sequences (Abstract and page 4). Fan et al. teach a library of random 10-nt sequences was inserted before the start codon of circRNA-enriched GFP, and the library was transfected into 293 T cells to generate circRNAs that can be translated into intact GFP, cells with the translated circRNA were recovered and then sequenced the inserted fragments to identify the IRES-like elements that drive circRNA translation, including comparing the resulting 10-mers to extract and identify hexamers that are significantly enriched in the cells with GFP fluorescence (Results, pages 5-6). Fan et al. teach that the enriched sequences are generally AU-rich, and the hexamers have strong dinucleotide biases toward AT, AG and GA (page 6, top). Fan et al. teach the IRES-like element hexamers were inserted into a circRNA reporter for translation of GFP (Figure 1E Legend, page 29). Therefore, Fan et al. teach a circular RNA having IRES-like hexamers inserted into an expression cassette for expressing a foreign protein (the GFP).
Fan et al. does not teach the nucleotide sequence of translation initiation element is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-40.
However, before the effective filing date, Guo et al. teach a need to develop a new DNA element that can enhance protein translation efficiency (paragraphs 0007,0009). Guo et al. teach a nucleic acid construct of formula II from 5’ to 3’: Z1-Z2-Z3-Z4-Z5 (II) wherein Z1,Z2,Z3,Z4 and Z5 are respectively elements used to constitute the construct, each “-“ is a bond or a nucleotide linking sequence; Z1 is the 5’ leading sequence of tobacco mosaic virus Ω sequence, Z2 is an oligomeric chain of adenine deoxynucleotides, Z3 is the translation initiation codon, Z4 is a serine codon, and Z5 is a coding sequence of the exogenous protein, and that Z2,Z3 AND Z4 constitute the Kozak sequence (paragraphs 0033-0043). Guo et al. teach the Kozak sequence used includes 6-12 adenine deoxynucleoside oligomer chains, a translation initiation codon (such as ATG, ATA, ATT, GTG, TTG, etc.) and a serine codon (such as TCT, TCC, TCA,TCG, AGT, AGC, etc.) and is derived from Kluveromyces (paragraph 0123). Guo et al. teach that the novel DNA element contained in the present invention has the potential for further optimization (paragraph 0167).
Guo et al. also teach a vector comprising the nucleic acid construct (paragraphs 0050,0152), and a genetically engineered cell with one or more sites of the genome integrated with the construct, including eukaryotic cells such as yeast cells, and prokaryotic cells including E. coli, Streptomyces, Agrobacterium, higher animal cells, insect cells (paragraphs 0051,0154,0157).
Guo et al. teach a method for high throughput in-vitro synthesis of exogenous proteins by providing the nucleic acid construct in the presence of a yeast in vitro protein synthesis system and incubating the yeast in vitro protein synthesis system for a period of time under suitable conditions thereby synthesizing the exogenous protein (paragraphs 0064,0160,0161). Guo et al. teach when the DNA element of the present invention is used in an in vitro yeast protein synthesis system, the relative light value of the synthesized luciferase activity is enhanced by approximately 22.3 times compared to a DNA element containing only the Ω sequence (paragraph 0164).
Additionally, Noderer et al. teach using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing start codons, and measured translation from a genetic reporter library (Abstract). Noderer et al. teach a genetic reporter system where different TIS sequences of interest were used to initiate translation of green fluorescent protein, and developed a TIS reporter library by inserting TIS sequences with randomly chosen bases flanking the AUG start codon at positions -6 to -1, +4 and +5 (i.e. NNNNNNAUGNN) (Results page 2, left column, and Fig 1A).
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Noderer et al. teach “with some optimization, we believe that FACS-seq can be applied to libraries approaching 106 sequences, with the practical limit being determined by the time required to sort the library. Our ability to analyze such as large number of sequences was dependent on the precision of the readout from our translation reporter….Massively parallel methods like FACS-seq enable the thorough analysis of a desired sequence space such that one can now predict the behavior of all sequence elements employed in the genome” (page 10, right column).
Noderer et al. also teach PD-31 mouse pre-B lymphocytes were transduced with the entire TIS reporter library (Page 2, right column).
Regarding claim 15, Fan et al. teach reporter circRNAs that encode GFP and the IRES-like elements have m6A sites in order to drive circRNA translation (Figure Legend, Figure 2B, page 30).
Regarding claim 16, Fan et al. teach a vector comprising the circular RNA comprising the hexamer inserted into the circRNA GFP reporter (Figure Legend: Fig 1E, page 29).
Regarding claims 17 and 18, Fan et al. teach the hexamers were inserted into the circRNA reporter containing GFP, which was transfected into 293T cells (Figure Legend: Fig 1E, page 29).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to optimize the nucleotide sequence used for translation initiation as taught by Fan et al., Guo et al. and Noderer et al. in prokaryotic to eukaryotic cell types, as optimization is required in order to increase the amounts of protein produced, to arrive at the circular RNA construct of the instant claims with a reasonable expectation of success. There would be a reasonable expectation of success, because Fan et al., Guo et al. and Noderer et al. all pertain to analyzing and optimizing sequences for translation initiation. One of ordinary skill in the art would be motivated to optimize the nucleotide sequence used as a translation initiation element in a circular RNA construct comprising a translation initiation element and an expression cassette comprising a nucleotide sequence encoding a foreign protein, because Fan et al. teach screening of a random 10-nt library using a cell-based reporter system and identified a large number of AU-rich motifs capable of initiating circRNA translation, and that these IRES-like elements are significantly enriched in circRNAs and are sufficient to drive robust translation of circRNAs. Additionally, Guo et al. teach a need to develop a new DNA element that can enhance protein translation efficiency and the Kozak sequence used includes 6-12 adenine deoxynucleoside oligomer chains, a translation initiation codon and a serine codon and that the novel DNA element contained in the present invention has the potential for further optimization. Therefore, Guo et al. teach the range of 6-12 adenines in the new DNA element to enhance protein translation and optimization thereof. In addition, one of ordinary skill in the art would also be motivated by the teachings of Noderer et al., using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing start codons, and measured translation from a genetic reporter library and that different TIS sequences of interest were used to initiate translation of green fluorescent protein, and developed a TIS reporter library by inserting TIS sequences with randomly chosen bases flanking the AUG start codon at positions -6 to -1, +4 and +5 (i.e. NNNNNNAUGNN). An ordinary artisan could use the teachings of Fan et al., Guo et al. and Noderer et al. regarding screening and analyzing of various lengths and sequences containing different numbers and positions of A’s that provide translation initiation to optimize the translation initiation element in the circular RNA construct to arrive at any of the translation initiation sequences of SEQ ID NOs: 1-40 of the instant claims.
Accordingly, the limitations of claims 11,12 and 14-18 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claims 19 and 20 are rejected under 35 U.S.C. 103 as being obvious over Fan et al. Guo et al., and Noderer et al. as applied to claims 11,12 and 14-18 and further in view of Yang et al. (Cell Research 27:626-641, Published 10 March 2017), cited on an IDS and Gregorio et al. (Methods Protoc., 12 March 2019, 2, 24, pages 1-34).
The teachings of Fan et al., Guo et al. and Noderer et al. as applicable to claims 11,12 and 14-18 are described above.
Guo et al. also teach a method for high throughput in-vitro synthesis of exogenous proteins by providing the nucleic acid construct in the presence of a yeast in vitro protein synthesis system and incubating the yeast in vitro protein synthesis system for a period of time under suitable conditions thereby synthesizing the exogenous protein (paragraphs 0064,0160,0161).
Fan et al., Guo et al. and Noderer et al. do not teach a reaction system that contains other components for the reaction selected from the group consisting of spliceosomes, ribosomes, translation initiation factor EIF4G2, translation initiation factor EIF4A, translation initiation factor EIF4B, and combinations thereof, or that the reaction system and method of synthesizing protein in vitro is cell-free.
However, before the effective filing date, Yang et al. teach circRNAs, vectors encoding a circular RNA construct (Figures 1A and 1C), and transfection of the circRNA into 293 cells (Figure 1B and page 629, left column). Yang et al. teach circRNA translation needs to be initiated through a mechanism fundamentally different from linear mRNA, and that eukaryotic translation is initiated by EIF4 complex (page 630, right column). Yang et al. teach the investigation of EIF4G2 in translation initiation of circRNAs, and found that translation of circRNA may be initiated by an EIF4G2-dependent mechanism similar to other IRESs (page 631, left column). Yang et al. teach that in order to further understand m6A-driven translation of circRNAs, the expression of GFP encoded by circRNA in stable cell lines was examined and found that EIF4G2 depletion significantly reduced protein translation from circRNA, but that overexpression of EIF4G2 increased GFP translation from circRNA by co-expressing EIF4G2 with the circRNA (page 631, left column). Therefore, Yang et al. teach the production of a protein (GFP) by incubating the translation initiation factor EIF4G2 with the circRNA.
Gregorio et al. teach cell-free protein synthesis (CFPS), which is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development and education, and provide advantages over in vivo protein expression including its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest (Abstract, page 1). Gregorio et al. teach that metabolic and cytotoxic burdens placed on the cell when trying to produce large quantities of recombinant proteins in vivo are obviated in CFPS, and the CFPS platform is amenable to direct manipulation of the environment of protein production because it is an open system, and in some cases high protein titers can be achieved (Page 2, first paragraph). Gregorio et al. also teach that the CFPS reactions are flexible in their setup, allowing users to utilize a variety of formats to achieve desired protein titer, making CFPS optimally suited for production of difficult-to-synthesize proteins, large proteins, proteins encoded by high GC content genes, membrane proteins and virus-like particles (page 2, first paragraph).
Gregorio et al. teach a variety of commercial CFPS kits are available, including cell-extract-based CFPS kits, and PURExpress kit which is comprised of a reconstitution of purified components of the transcription and translation machinery from E coli, which uses 10 translation factors including ribosomes (Section 2.1, page 4). Gregorio et al. teach CFPS reactions are most easily, quickly and cheaply set up in a batch format because all necessary reactants are added to a single tube and incubated for protein synthesis to occur (Section 2.3, page 4).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to provide the optimized circular RNA construct of Fan et al. Guo et al. and Noderer et al. in an in vitro cell-free reaction system with the translation initiation factor EIF4G2 with a reasonable expectation of success. There would be a reasonable expectation of success as Yang et al. also pertains to circRNAs encoding for GFP and translation initiation, and Gregorio et al. teaches cell-free translation machinery for cell-free protein synthesis. One of ordinary skill in the art would have been motivated to provide the translation initiation factor EIF4G2 in a reaction system with the optimized circRNA of Fan et al., Guo et al. and Noderer et al., because Yang et al. teach circRNA translation needs to be initiated through a mechanism fundamentally different from linear mRNA, and that eukaryotic translation is initiated by EIF4 complex (page 630, right column), and found that translation of circRNA may be initiated by an EIF4G2-dependent mechanism similar to other IRESs (page 631, left column). Yang et al. teach overexpression of EIF4G2 increased GFP translation from circRNA by co-expressing EIF4G2 with the circRNA (page 631, left column). One of ordinary skill in the art would have been motivated to provide a cell-free in vitro reaction system comprising the optimized circular RNA construct of Fan et al. Guo et al. and Noderer et al. and translation initiation factor EIF4G2 of Yang et al. because Gregorio et al. teach there are many benefits to using cell-free protein synthesis including its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest, and production of high protein titers, as well as the availability of a variety of commercial CFPS kits such as the PURExpress kit which is comprised of a reconstitution of purified components of the transcription and translation machinery from E coli, which uses 10 translation factors including ribosomes. Accordingly, the limitations of claim 19 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
It would have been obvious to one of ordinary skill in the art before the effective filing date, to provide a method for synthesizing protein in vitro using a cell-free reaction system by incubating the reaction system comprising the optimized circular RNA construct of Fan et al., Guo et al. and Noderer et al. with the EIF4G2 translation initiation factor of Yang et al. based on the protein synthesis method of Guo et al. and cell free protein synthesis taught by Gregorio et al. with a reasonable expectation of success. There would be a reasonable expectation of success as Fan et al. and Yang et al. both pertain to circRNA and Guo et al. teach a method for high throughput in-vitro synthesis of exogenous proteins by providing the nucleic acid construct in the presence of a yeast in vitro protein synthesis system and incubating the yeast in vitro protein synthesis system for a period of time under suitable conditions thereby synthesizing the exogenous protein (paragraphs 0064,0160,0161), and Gregorio et al. teach cell-free translation machinery for cell-free protein synthesis. One of ordinary skill in the art would be motived to provide the translation initiation factor EIF4G2 in a reaction system used in the protein synthesis method with the optimized circRNA of Fan et al., Guo et al. and Noderer et al., because Yang et al. teach overexpression of EIF4G2 increased GFP translation from circRNA by co-expressing EIF4G2 with the circRNA (page 631, left column). One of ordinary skill in the art would have been motivated to provide a cell-free in vitro method for synthesizing protein because Gregorio et al. teach there are many benefits to using cell-free protein synthesis including its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest, and production of high protein titers, as well as the availability of a variety of commercial CFPS kits such as the PURExpress kit which is comprised of a reconstitution of purified components of the transcription and translation machinery from E coli, which uses 10 translation factors including ribosomes. Accordingly, the limitations of claim 19 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., Guo et al., Noderer et al., Yang et al. and Gregorio et al. as applied to claims 19 and 20 above, and further in view of US 20170298347 (Pandolfi et al.), Published 19 October 2017.
Claim Interpretation: The preamble of claim 21 recites “A kit for synthesizing protein in vitro”, however this is just an intended use and is not being considered a claim limitation.
The teachings Fan et al., Guo et al., Noderer et al., Yang et al. and Gregorio et al. as applicable to claims 19 and 20 are described above.
Additionally, Gregorio et al. teach different cell-free protein synthesis platforms that use different vessels and conditions in Table 3 on page 23. Gregorio et al. teach the E. coli cell free protein synthesis platform can use many reaction vessels and that the yield increases as the surface area to reaction volume ratio increases (Table 3, page 23).
Fan et al., Guo et al., Noderer et al., Yang et al. and Gregorio et al. do not teach that the kit for synthesizing a protein in vitro comprising a first container comprising the construct of claim 11, a second container containing other components required for the synthesizing reaction, and a label or instructions for using the kit.
However, before the effective filing date, Pandolfi et al. teach novel fusion-circular RNAs and complements thereof [0003]. Pandolfi et al. teach a kit including a suitable container containing a nucleic acid sequence (e.g. an expression vector) encoding an f-circRNA or complement thereof and optional reagents for use with the vector, and it may be desirable to provide the components of the kit separately in two or more containers (one container for the expression vector and another for other reagents), and the components can be combined according to instructions provided with the kit [0343].
Therefore, it would have been obvious to a person of ordinary skill in the art at the time of the effective filing date, to provide the optimized circRNA construct of Fan et al., Guo et al. and Noderer et al. and translation initiation factor, EIF4G2 of Yang et al. in an in vitro cell free kit for protein synthesis as taught by Gregorio et al., and to provide multiple containers, one for each of the circRNA construct and the EIF4G2 translation initiation factor, and instructions for using the kit based on the teachings of Pandolfi et al., with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so in order to keep the components (circRNA construct and translation initiation factor(s) separate until the time needed and to combine the components according to the provided instructions as taught by Pandolfi et al. [0343] to use for in vitro cell-free protein synthesis as taught by Gregorio et al.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Conclusion
Claims 11,12 and 14-21 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEPHANIE L SULLIVAN whose telephone number is (703)756-4671. The examiner can normally be reached Monday-Friday, 7:30-3:30 EST.
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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635