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 .
Applicant’s election without traverse of SEQ ID NO:33948 as the IRES and SEQ ID NO: 28977 as the sequence that is complementary to an 18S rRNA in the reply filed on 4/29/25 is acknowledged.
Claim Rejections - 35 USC § 112
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 42-74 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.
Claim 42 is directed to a polynucleotide comprising a recombinant circular RNA molecule, wherein the recombinant circular RNA molecule comprises a protein-coding nucleic acid sequence and an internal ribosome entry site (IRES) sequence region operably linked to the protein-coding nucleic acid sequence, wherein the IRES sequence region comprises at least one RNA secondary structure element, wherein the RNA secondary structure element is a Stem-loop Structured RNA Element (SuRE), and wherein the first nucleic acid at the 5' end of the IRES is considered to be position 1. The specification does not adequately describe the genus of polynucleotides comprising a recombinant circular RNA molecule, wherein the recombinant circular RNA molecule comprises any protein-coding sequence and wherein the IRES region comprises any SuRE that functions as required.
Claim 43 is directed to a polynucleotide comprising a recombinant circular RNA molecule, wherein the recombinant circular RNA molecule comprises a protein-coding nucleic acid sequence and an internal ribosome entry site (IRES) sequence region operably linked to the protein-coding nucleic acid sequence, wherein the IRES sequence region has a GC content of greater than 25%. The specification does not adequately describe the genus of polynucleotides comprising a recombinant circular RNA molecule, wherein the recombinant circular RNA molecule comprises any protein-coding nucleic acid sequence and any internal ribosome entry site (IRES) sequence region operably linked to the protein-coding nucleic acid sequence, wherein the IRES sequence region has a GC content of any percentage greater than 25% and has the required function.
Claim 44 is directed to a polynucleotide comprising a recombinant circular RNA molecule that comprises a protein-coding nucleic acid sequence and an IRES sequence region operably linked to the protein-coding nucleic acid, wherein the IRES sequence region comprises at least one sequence region of any length (i.e. 2 nucleotides) that is complementary to an 18S ribosomal RNA.
The specification discloses identification of 18S rRNA complementary sequences that facilitate circRNA translation. The specification discloses specific sequences that were identified as active regions on 18S rRNA that complementary sequences in circRNA IRES can facilitate cap-independent translation on circRNA (Example 3).
However, the instant claims are not limited to regions of any specific length that are complementary to any specific 18S rRNA sequence that would render the instant polynucleotide functional.
The specification discloses that stem-loop structures at distinct positions on circRNA IRES facilitates cap-independent translation (Example 5). When the stem-loop was disrupted or moved, translation was decreased.
However, the claims are not limited to stem-loop structures at any specific position of any specific polynucleotide type. The breadth of the claims is much broader than the structural criteria of the specification required for the function. The species of the specification are not representative of the entire claimed genus.
The specification discloses that the results along with the 18S rRNA profiling suggest that two key regulatory elements on circRNA IRES, the 18S rRNA complementarity and 40-60 nt SuRE on the IRES, can facilitate cap-independent translation on circRNA [0114] and are important for activity [0116].
The specification discloses: [0135] Interestingly, while the circFGFR1 IRES (oligo index: 8228) displayed strong cap- independent translation activity on the circRNA (top 2%), the same IRES showed very weak cap- independent translation activity on the linear RNA (bottom 10%) (Weingarten-Gabbay et al., 2016). This observation suggests that the cap-independent translation activity of circFGFR1 IRES is preferentially activated on circFGFR1 rather than the linear FGFR1 transcripts.
However, the claims are directed to polynucleotides comprising a sequence coding any possible protein, wherein the polynucleotide IRES sequence comprises any region of any length that is complementary to any region of any 18S ribosomal RNA.
None of the instant claims require each of the structural requirements described in the specification as being mandatory for function.
Instant claim 56 requires for the length of the spacer to be selected to increase translation of the protein-coding nucleic acid sequence region relative to translation of a circular RNA having no spacer. However, the specification does not adequately describe the structure required for the function. The specification does not adequately describe what length of spacer is needed to result in increased translation.
Instant claim 57 requires for the IRES sequence region to be configured to promote rolling circle translation. However, the specification does not adequately describe the structure required for the function. The specification does not adequately describe what configuration is required for the IRES sequence region to promote rolling circle translation.
The MPEP states that for a generic claim, the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. See MPEP § 2163. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. See MPEP § 2163. Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad genus. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618. Additionally, in Carnegie Mellon University v. Hoffman-La Roche Inc., Nos. 07-1266, -1267 (Fed. Cir. Sept. 8, 2008), the Federal Circuit affirmed that a claim to a genus described in functional terms was not supported by the specification’s disclosure of species that were not representative of the entire genus. Furthermore, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
The claims are rejected under the written description requirement for failing to disclose adequate species to represent the claimed genus that would function as required.
The Guidelines for Examination of Patent Applications under the 35 USC § 112, first paragraph, “Written Description” Requirement”, published at Federal Register, Vol. 66, No. 4, pp. 1099-1111 outline the method of analysis of claims to determine whether adequate written description is present. The first step is to determine what the claim as a whole covers, i.e., discussion of the full scope of the claim. Second, the application should be fully reviewed to understand how applicant provides support for the claimed invention including each element and/or step, i.e., compare the scope of the claim with the scope of the description. Third, determine whether the applicant was in possession of the claimed invention as a whole at the time of filing.
To achieve the desired function, it appears that the structure is required to comprise additional elements. Kruse (WO 2014/186334 A1) teaches that a circular mRNA possesses additional RNA elements compared to containing only an IRES element for successful in vitro or in vivo translation (abstract). Kruse et al. teach that: [0003] The disclosure generally relates to a biologic product comprising a circular RNA that is capable of translation inside a eukaryotic cell. The invention describes novel combinations of RNA elements that facilitate the enhanced translation and expression of encoded polypeptides, and provides vectors for making circular mRNA, as well as various applications using the circular mRNA and/or vector.
Kruse et al. teach: [0010] Thus, the complete mRNA in the current art needs a 5' cap or cap analogue, 5' UTR, ORF, 3' UTR, and polyadenylation tail to mimic the standard mRNA molecule produced by eukaryotic cells. In some cases, a 5' cap is omitted and an IRES sequence utilized, but this is much more inefficient and reduces the half-life of the linear RNA molecule with no protection of the 5' terminus of RNA.
Therefore, it is clear that additional structural requirements are needed in addition to an IRES linked to a protein-coding sequence, wherein the IRES comprises any possible SuRE or any sized region complementary to any region of any 18S rRNA or any IRES sequence having any percentage over 25% of GC content, for the compound to function as required.
Thus, having analyzed the claims with regard to the Written Description guidelines, it is clear that the specification does not disclose a representative number of species to adequately describe the entire claimed genuses. Thus, one skilled in the art would be led to conclude that Applicant was not in possession of the claimed invention at the time the application was filed.
Claim Rejections - 35 USC § 102
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 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.
Claim(s) 43, 60, 64, and 65 is/are rejected under 35 U.S.C. 102(a)(1) or (a)(2) as being anticipated by Kruse (WO 2014/186334 A1).
Kruse et al. teach: A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation (abstract).
Kruse et al. teach that the circular mRNA comprises the IRES and open reading frame, wherein the elements are operably linked (Novelty).
[0003] The disclosure generally relates to a biologic product comprising a circular RNA that is capable of translation inside a eukaryotic cell. The invention describes novel combinations of RNA elements that facilitate the enhanced translation and expression of encoded polypeptides, and provides vectors for making circular mRNA, as well as various applications using the circular mRNA and/or vector.
[0010] Thus, the complete mRNA in the current art needs a 5' cap or cap analogue, 5' UTR, ORF, 3' UTR, and polyadenylation tail to mimic the standard mRNA molecule produced by eukaryotic cells. In some cases, a 5' cap is omitted and an IRES sequence utilized, but this is much more inefficient and reduces the half-life of the linear RNA molecule with no protection of the 5' terminus of RNA.
[0022] The circular mRNA can be transfected as is, or can be transfected in DNA vector form and transcribed in the cell, as desired. Cellular transcription can use added polymerases or nucleic acids encoding same, or preferably can use endogenous polymerases. We have demonstrated proof of concept herein with added T7 polymerases, but this is exemplary only, and more convenient cell based polymerases may be preferred. [0023] The preferred half-life of a circular mRNA in a eukaryotic cell is at least 20 hrs, 30 hrs or even at least 40 hrs, as measured by either a hybridization or quantitative RT-PCR experiments.
[0024] A preferred embodiment of the invention consists of a circular mRNA molecule with an IRES, 5' UTR, coding sequence of interest, 3' UTR and polyadenylation sequence, in that order. It is well appreciated that many different combinations of these RNA elements with translation enhancing properties and synergy can be created. Such combinations include but are not limited to IRES-ORF-3' UTR polyA, IRES-ORF-3' UTR, IRES-5' UTR-ORF-3' UTR, and the like.
[0087] Beyond utilizing novel IRES sequences, adding other RNA elements to the circular mRNA molecule allow for translation inside cells. It is readily recognized for example, that while the cap is an important structure for eukaryotic linear mRNA translation, the 5' UTR, 3' UTR and polyA tails also play important roles in translation.
[0088] The preferred embodiment of the invention contains a polyadenylation sequence within the circular RNA molecule of about 30-ribonucleotides of adenosine, which is able to bind to a single complex of human poly(A)-binding protein. This 20 polyadenylation sequence would be located after the ORF, 3'UTR and before the splice site and termination signal.
[0089] Polyadenylation of mRNAs have been shown to increase the expression of viral IRES driven expression.
[0093] In another embodiment of the invention, a 5' UTR will be utilized that will facilitate the delivery of the ribosome to the first codon of the polypeptide to be translated. The mechanism of ribosomal tethering and delivery to downstream AUG codons would also be useful in circular mRNA molecules. This process is also referred to "ribosomal shunting." An example of a sequence that mediates shunting is an mRNA element from the 5' UTR of the Gtx homodomain mRNA, which basepairs to 18S rRNA, and the adenovirus tripartite leader.
Kruse et al. teach that the IRES can comprise SEQ ID NO: 3, which has 52% GC content (45/86 nucleotides).
Kruse et al. tach a vector for production of the circRNA.
Therefore, Kruse et al. teach a circular mRNA comprising a protein-coding sequence that is operably linked to an IRES region, wherein the GC content of the IRES sequence is greater than 25% (instant claim 43).
Kruse et al. teach: [0056] By "vector" or "cloning vector" what is meant is a small piece of DNA, taken from a virus, plasmid, or cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning and/or expression purposes. A vector typically has an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and usually contains a multiple cloning site. The term includes plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like (instant claim 60).
Kruse et al. teach: [0026] Another embodiment of the invention consists of the in vitro transcription of a DNA template encoding the circular mRNA molecule of interest. Inverted intron self-splicing sequences at both ends of the RNA molecule facilitate the formation of circular RNA without any additional enzymes being needed.
Kruse et al. teach: [0098] The technologies required to produce circular RNA have been described in the literature previously. Commonly, group I self-splicing by a permuted intron-exon sequences from the T4 bacteriophage is used. This reaction can occur in prokaryotic cells, eukaryotic cells, or in vitro since it is catalyzed by RNA alone. However, a variety of different methods exist in that prior art concerning ways to synthesize circular RNA. It is understood that the proposed enhanced circular mRNA molecule could use any of these methods in its production (e.g., US6210931, US5773244). [0099] Examples of group I intron self-splicing sequences include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td. The intervening sequence (IVS) rR A of Tetrahymena also contains an example of a Group I intron self-splicing sequences. Given the widespread existence of group I and group II catalytic introns across nature, many possible sequences could be used for creating circular RNA (instant claims 64 and 65).
Therefore, the claims are anticipated by Kruse et al.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 43 and 60-66 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kruse (WO 2014/186334 A1), as applied to claims 43, 60, 64, and 65 above, and further in view of Anderson et al. (WO 2019/236673 A1), Cerrato et al. (BioMol Concepts 2014; 5(6): 479–488), and Patop et al. (The EMBO Journal 38: e100836, 2019, 1-13).
Kruse et al. teach: A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation (abstract).
Kruse et al. teach that the circular mRNA comprises the IRES and open reading frame, wherein the elements are operably linked (Novelty).
[0003] The disclosure generally relates to a biologic product comprising a circular RNA that is capable of translation inside a eukaryotic cell. The invention describes novel combinations of RNA elements that facilitate the enhanced translation and expression of encoded polypeptides, and provides vectors for making circular mRNA, as well as various applications using the circular mRNA and/or vector.
[0010] Thus, the complete mRNA in the current art needs a 5' cap or cap analogue, 5' UTR, ORF, 3' UTR, and polyadenylation tail to mimic the standard mRNA molecule produced by eukaryotic cells. In some cases, a 5' cap is omitted and an IRES sequence utilized, but this is much more inefficient and reduces the half-life of the linear RNA molecule with no protection of the 5' terminus of RNA.
[0022] The circular mRNA can be transfected as is, or can be transfected in DNA vector form and transcribed in the cell, as desired. Cellular transcription can use added polymerases or nucleic acids encoding same, or preferably can use endogenous polymerases. We have demonstrated proof of concept herein with added T7 polymerases, but this is exemplary only, and more convenient cell based polymerases may be preferred. [0023] The preferred half-life of a circular mRNA in a eukaryotic cell is at least 20 hrs, 30 hrs or even at least 40 hrs, as measured by either a hybridization or quantitative RT-PCR experiments.
[0024] A preferred embodiment of the invention consists of a circular mRNA molecule with an IRES, 5' UTR, coding sequence of interest, 3' UTR and polyadenylation sequence, in that order. It is well appreciated that many different combinations of these RNA elements with translation enhancing properties and synergy can be created. Such combinations include but are not limited to IRES-ORF-3' UTR polyA, IRES-ORF-3' UTR, IRES-5' UTR-ORF-3' UTR, and the like.
[0087] Beyond utilizing novel IRES sequences, adding other RNA elements to the circular mRNA molecule allow for translation inside cells. It is readily recognized for example, that while the cap is an important structure for eukaryotic linear mRNA translation, the 5' UTR, 3' UTR and polyA tails also play important roles in translation.
[0088] The preferred embodiment of the invention contains a polyadenylation sequence within the circular RNA molecule of about 30-ribonucleotides of adenosine, which is able to bind to a single complex of human poly(A)-binding protein. This 20 polyadenylation sequence would be located after the ORF, 3'UTR and before the splice site and termination signal.
[0089] Polyadenylation of mRNAs have been shown to increase the expression of viral IRES driven expression.
[0093] In another embodiment of the invention, a 5' UTR will be utilized that will facilitate the delivery of the ribosome to the first codon of the polypeptide to be translated. The mechanism of ribosomal tethering and delivery to downstream AUG codons would also be useful in circular mRNA molecules. This process is also referred to "ribosomal shunting." An example of a sequence that mediates shunting is an mRNA element from the 5' UTR of the Gtx homodomain mRNA, which basepairs to 18S rRNA, and the adenovirus tripartite leader.
Kruse et al. teach that the IRES can comprise SEQ ID NO: 3, which has 52% GC content (45/86 nucleotides).
Kruse et al. tach a vector for production of the circRNA.
Therefore, Kruse et al. teach a circular mRNA comprising a protein-coding sequence that is operably linked to an IRES region, wherein the GC content of the IRES sequence is greater than 25% (instant claim 43).
Kruse et al. teach: [0056] By "vector" or "cloning vector" what is meant is a small piece of DNA, taken from a virus, plasmid, or cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning and/or expression purposes. A vector typically has an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and usually contains a multiple cloning site. The term includes plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like (instant claim 60).
Kruse et al. teach: [0026] Another embodiment of the invention consists of the in vitro transcription of a DNA template encoding the circular mRNA molecule of interest. Inverted intron self-splicing sequences at both ends of the RNA molecule facilitate the formation of circular RNA without any additional enzymes being needed.
Kruse et al. teach: [0098] The technologies required to produce circular RNA have been described in the literature previously. Commonly, group I self-splicing by a permuted intron-exon sequences from the T4 bacteriophage is used. This reaction can occur in prokaryotic cells, eukaryotic cells, or in vitro since it is catalyzed by RNA alone. However, a variety of different methods exist in that prior art concerning ways to synthesize circular RNA. It is understood that the proposed enhanced circular mRNA molecule could use any of these methods in its production (e.g., US6210931, US5773244). [0099] Examples of group I intron self-splicing sequences include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td. The intervening sequence (IVS) rR A of Tetrahymena also contains an example of a Group I intron self-splicing sequences. Given the widespread existence of group I and group II catalytic introns across nature, many possible sequences could be used for creating circular RNA (instant claims 64 and 65).
Kruse et al. does not teach incorporation into a lipid nanoparticle that is coupled to a targeting agent.
However, it was known to formulate circular RNAs into nanoparticles and to incorporate targeting agents as claimed, as evidenced by Anderson et al. and Cerrato et al. It would have been obvious to incorporate these elements as a matter of design choice as nanoparticles with targeting agents are routine in the nucleic acid field. G-C content is considered routine in the art and a matter of design choice.
Anderson et al. teach that the purified circular RNA is formulated into nanoparticles and is compatible with nanoparticles. The nanocarrier is a lipid, polymer or a lipo-polymeric hybrid. Anderson et al. teach: A vector for making circular RNA, comprising the following elements operably connected to each other and arranged in the following sequence: (a) a 5' homology arm, (b) a 3' group I intron fragment containing a 3' splice site dinucleotide, (c) a 5' spacer sequence, (d) an internal ribosome entry site (IRES), (e) a protein coding region or noncoding region, (f) a 3' spacer sequence, (g) a 5' group I intron fragment containing a 5' splice site dinucleotide, and (h) a 3' homology arm, is new. The vector allows production of circular RNA that is translatable or biologically active inside eukaryotic cells.
Cerrato et al. teach that peptides and peptide-cargo complexes have been used for drug delivery and gene therapy. One of the most used delivery vectors are cell-penetrating peptides, due to their ability to be taken up by a variety of cell types and deliver a large variety of cargoes through the cell membrane with low cytotoxicity (abstract). Cerrato et al. teach that the peptide is coupled to a nanoparticle (pages 483 and 484 (instant claims 61-63).
Kruse et al. do not teach incorporation of a miRNA binding site or RBP binding site. However, it was known to incorporate miRNA binding sites into circRNAs, as taught by Patop et al. (page 5). Patop et al. teach that circRNAs can comprise binding sites for RBPs and miRNAs (page 6). Incorporation of a miRNA binding site is considered to be a matter of design choice.
Claim(s) 42-53, 55-69, and 71-74 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kruse (WO 2014/186334 A1), in view of Kieft et al. (Methods in Enzymology, Volume 430, 333-371), Atabekov et al. (WO 03/020928 A2), Asokan et al. (WO 2019/094486 A1), Anderson et al. (WO 2019/236673 A1), Cerrato et al. (BioMol Concepts 2014; 5(6): 479–488), and Patop et al. (The EMBO Journal 38: e100836, 2019, 1-13).
Kruse et al. teach: A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation (abstract).
Kruse et al. teach that the circular mRNA comprises the IRES and open reading frame, wherein the elements are operably linked (Novelty).
[0003] The disclosure generally relates to a biologic product comprising a circular RNA that is capable of translation inside a eukaryotic cell. The invention describes novel combinations of RNA elements that facilitate the enhanced translation and expression of encoded polypeptides, and provides vectors for making circular mRNA, as well as various applications using the circular mRNA and/or vector.
[0010] Thus, the complete mRNA in the current art needs a 5' cap or cap analogue, 5' UTR, ORF, 3' UTR, and polyadenylation tail to mimic the standard mRNA molecule produced by eukaryotic cells. In some cases, a 5' cap is omitted and an IRES sequence utilized, but this is much more inefficient and reduces the half-life of the linear RNA molecule with no protection of the 5' terminus of RNA.
[0022] The circular mRNA can be transfected as is, or can be transfected in DNA vector form and transcribed in the cell, as desired. Cellular transcription can use added polymerases or nucleic acids encoding same, or preferably can use endogenous polymerases. We have demonstrated proof of concept herein with added T7 polymerases, but this is exemplary only, and more convenient cell based polymerases may be preferred. [0023] The preferred half-life of a circular mRNA in a eukaryotic cell is at least 20 hrs, 30 hrs or even at least 40 hrs, as measured by either a hybridization or quantitative RT-PCR experiments.
[0024] A preferred embodiment of the invention consists of a circular mRNA molecule with an IRES, 5' UTR, coding sequence of interest, 3' UTR and polyadenylation sequence, in that order. It is well appreciated that many different combinations of these RNA elements with translation enhancing properties and synergy can be created. Such combinations include but are not limited to IRES-ORF-3' UTR polyA, IRES-ORF-3' UTR, IRES-5' UTR-ORF-3' UTR, and the like.
[0087] Beyond utilizing novel IRES sequences, adding other RNA elements to the circular mRNA molecule allow for translation inside cells. It is readily recognized for example, that while the cap is an important structure for eukaryotic linear mRNA translation, the 5' UTR, 3' UTR and polyA tails also play important roles in translation.
[0088] The preferred embodiment of the invention contains a polyadenylation sequence within the circular RNA molecule of about 30-ribonucleotides of adenosine, which is able to bind to a single complex of human poly(A)-binding protein. This 20 polyadenylation sequence would be located after the ORF, 3'UTR and before the splice site and termination signal.
[0089] Polyadenylation of mRNAs have been shown to increase the expression of viral IRES driven expression.
[0093] In another embodiment of the invention, a 5' UTR will be utilized that will facilitate the delivery of the ribosome to the first codon of the polypeptide to be translated. The mechanism of ribosomal tethering and delivery to downstream AUG codons would also be useful in circular mRNA molecules. This process is also referred to "ribosomal shunting." An example of a sequence that mediates shunting is an mRNA element from the 5' UTR of the Gtx homodomain mRNA, which basepairs to 18S rRNA, and the adenovirus tripartite leader.
Kruse et al. teach that the IRES can comprise SEQ ID NO: 3, which has 52% GC content (45/86 nucleotides). The length of the IRES sequence is considered to be a matter of design choice (instant claim 51).
Kruse et al. tach a vector for production of the circRNA.
Therefore, Kruse et al. teach a circular mRNA comprising a protein-coding sequence that is operably linked to an IRES region, wherein the GC content of the IRES sequence is greater than 25% (instant claim 43).
Kruse et al. teach: [0056] By "vector" or "cloning vector" what is meant is a small piece of DNA, taken from a virus, plasmid, or cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning and/or expression purposes. A vector typically has an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and usually contains a multiple cloning site. The term includes plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like (instant claim 60).
Kruse et al. teach: [0026] Another embodiment of the invention consists of the in vitro transcription of a DNA template encoding the circular mRNA molecule of interest. Inverted intron self-splicing sequences at both ends of the RNA molecule facilitate the formation of circular RNA without any additional enzymes being needed.
Kruse et al. teach: [0098] The technologies required to produce circular RNA have been described in the literature previously. Commonly, group I self-splicing by a permuted intron-exon sequences from the T4 bacteriophage is used. This reaction can occur in prokaryotic cells, eukaryotic cells, or in vitro since it is catalyzed by RNA alone. However, a variety of different methods exist in that prior art concerning ways to synthesize circular RNA. It is understood that the proposed enhanced circular mRNA molecule could use any of these methods in its production (e.g., US6210931, US5773244). [0099] Examples of group I intron self-splicing sequences include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td. The intervening sequence (IVS) rR A of Tetrahymena also contains an example of a Group I intron self-splicing sequences. Given the widespread existence of group I and group II catalytic introns across nature, many possible sequences could be used for creating circular RNA (instant claims 64 and 65).
Kruse et al. do not teach that the IRES sequence has a Stem-loop structured RNA element (instant claim 42) or wherein the IRES region has a sequence complementary to an 18S rRNA (instant claim 44).
However, Kieft et al. teach that IRES RNAs tend to have stem loops that are translation machinery recognition sites (Fig. 13.2 and page 361). The stem loop is present at about positions 40-60 (see Figure 13.2) and the sequence comprises a spacer. This meets the instant limitation of a SuRE, which is not defined in the specification as being directed to anything more than a RNA stem loop.
Atabekov et al. teach that a role of direct IRES RNA/18S rRNA interaction has been shown. The Gtx IRES contains several nonoverlapping segments having complementarity to 18S rRNA that were shown to mediate internal initiation of translation. Within one of these segments, a 9-nt GC-rich sequence CCGGCGGGU which is 100% complementary to 18S rRNA at nucleotides 1132-1124 was identified (instant claims 67 and 68). It was shown that synthetic IRESes composed of multiple linked copies of this 9-nt IRES module increased internal initiation dramatically in animal cells. Therefore, it would have been obvious to incorporate a sequence into the IRES that is complementary to 18s rRNA with an expectation of to mediate internal initiation of translation.
Asokan et al. teaches that in vitro transcription of a DNA template encoding the circular RNA molecule of this invention is achieved by the presence of inverted intron self-splicing sequences at both ends of the RNA molecule facilitate the formation of circular RNA without any additional enzymes being needed.
Additionally, Asokan et al. teach circRNAs comprising IRES and sequence complementary to 18S rRNA (Figures 2A-2E). Asokan et al. teach delivery from an AAV vector.
With regards to lipid nanoparticles, it was known to formulate circular RNAs into nanoparticles and to incorporate targeting agents as claimed, as evidenced by Anderson et al. It would have been obvious to incorporate these elements as a matter of design choice as nanoparticles with targeting agents are routine in the nucleic acid field. G-C content is considered routine in the art and a matter of design choice.
Anderson et al. teach that the purified circular RNA is formulated into nanoparticles. The nanocarrier is a lipid, polymer or a lipo-polymeric hybrid. Anderson et al. teach: A vector for making circular RNA, comprising the following elements operably connected to each other and arranged in the following sequence: (a) a 5' homology arm, (b) a 3' group I intron fragment containing a 3' splice site dinucleotide, (c) a 5' spacer sequence, (d) an internal ribosome entry site (IRES), (e) a protein coding region or noncoding region, (f) a 3' spacer sequence, (g) a 5' group I intron fragment containing a 5' splice site dinucleotide, and (h) a 3' homology arm, is new. The vector allows production of circular RNA that is translatable or biologically active inside eukaryotic cells.
Cerrato et al. teach that peptides and peptide-cargo complexes have been used for drug delivery and gene therapy. One of the most used delivery vectors are cell-penetrating peptides, due to their ability to be taken up by a variety of cell types and deliver a large variety of cargoes through the cell membrane with low cytotoxicity (abstract). Cerrato et al. teach that the peptide is coupled to a nanoparticle (pages 483 and 484 (instant claims 61-63).
Kruse et al. do not teach incorporation of a miRNA binding site or RBP binding site. However, it was known to incorporate miRNA binding sites into circRNAs, as taught by Patop et al. (page 5). Patop et al. teach that circRNAs can comprise binding sites for RBPs and miRNAs (page 6). Incorporation of a miRNA binding site is considered to be a matter of design choice. Selection of the specific miRNA is considered to be a matter of design choice depending upon the desired outcome (instant claim 59).
It is noted that instant claim 57 required for the IRES region to be configured to promote rolling circle translation. However, the claim does not recite any specific structural limitation to result in the intended outcome. The intended outcome is considered to necessarily flow from the polynucleotide in absence of recitation of an additional structural limitation.
Instant claim 56 recites that the length of the spacer is selected to increase translation of the protein-coding nucleic acid sequence, but does not recite any specific length required and therefore the claim is considered to be anticipated by the presence of a linker.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 42-74 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 11,560,567 B2. Although the claims at issue are not identical, they are not patentably distinct from each other because the claims of US ‘567 are directed to a recombinant circular RNA molecule comprising a protein-coding nucleic acid sequence and an IRES sequence region operably linked to the protein-coding sequence, wherein the IRES sequence region comprises at least one RNA secondary structure element and a sequence that is complementary to an 18S rRNA, which are elements required by the instant claims. US ‘567 recites that the secondary structure is at the same position as instantly claimed and the claim sets recite overlapping size limitations for the IRES. The specification of US ‘567 defines the secondary structure as including stem loop structured RNA elements and the polynucleotide being comprised within a viral vector. The claims are obvious variations of each other.
The species are obvious variations of the patented genus and the species anticipates the genus covering it.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/AMY ROSE HUDSON/Primary Examiner, Art Unit 1636