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 .
Election
In the response received on June 8, 2026, Applicant added new claims 16-18, and Applicant elects without traverse to prosecute Group II, claims 1, 3, 6-8 (Resp 6). Newly added claims 16-18 are directed to the non-elected inventions of Groups III (for claims 16 and 17) and Group I (claim 18). After examining, the Examiner determined that claim 14 (Group IX) which requires SEQ ID NO: 19 belongs with the elected Group II because it includes an N-terminal fusion, therefore the restriction requirement between Group II and Group IX is WITHDRAWN. Claims 1- 18 are pending, claims 2, 4, 5, 9-13, and 15-18 are withdrawn for being directed to non-elected inventions, and claims 1, 3, 6-8, and 14 are examined in this Office Action.
Applicant further elects the sequences of SEQ ID NOs: 6 and 14 in response to a requirement for species election (Resp 6). The invention of Group III was not elected, therefore SEQ ID NO: 14 was not examined. However, if the linking claim becomes allowable, then all linked claims will be rejoined and the sequences recited in the linked claims will be examined.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
A certified copy of CN 202111039979.X was received from the WIPO office, however it is entirely in Chinese.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Currently, the Examiner is examining as if the priority date is Sept. 6, 2022, which is the date the parent PCT application was filed.
Specification
The disclosure is objected to because of the following informalities: The acronym “M-MLV” appears with no definition. Whenever an acronym is used, the first time it appears it should be accompanied by the full terminology.
Appropriate correction is requested.
Sequence Compliance
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 C.F.R. § 1.821(a)(1) and (a)(2). However, this application fails to comply with the requirements of 37 C.F.R. §§ 1.821-1.825. The polypeptide “GGGGS” appears with no sequence identifier (Spec 9, line 34). It does not appear that the current sequence listing includes this polypeptide sequence in the sequence listing. Applicant must comply with the requirements of the sequence rules (37 CFR 1.821 - 1.825) by submitting an updated version of their sequence listing that includes an identifier for this polypeptide and by amending the Specification to insert the new sequence identifier in parentheses next to the disclosure of this polypeptide wherever it appears within the application.
Claim Interpretation
Claim 7 recites a sequence using the indefinite article “an”: “an amino acid sequence of SEQ ID NO: 6” (emphasis added). The use of an indefinite article renders the claim inclusive of small fragments of SEQ ID NO: 6, as small as di-peptide fragments. Claim 14 is interpreted the same way for “an amino acid sequence of SEQ ID NO: 19” (emphasis added).
Claim Objections
Claim 6 is objected to because of the following informalities: it is missing an article in front of “N-terminus or C-terminus”. Applicant is advised to insert - - the - - in between “fused at” and “N-terminus”. Appropriate correction is requested.
Claim Rejections - 35 USC § 112
Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 3, 6-8, and 14 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim.
Claim 3 recites “M-MLV reverse transcriptase or a functional variant thereof”, and it is unclear what is required to be a “functional variant thereof”.
The M-MLV reverse transcriptase has three different activities: RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and Ribonuclease H activity. To be considered a “functional variant thereof” would the variant need to have all three activities? Or just one of the activities? Would the variant need to have the same level of activity? It is unclear what would be required for the recited “functional variant”.
Claim 6 depends from claim 3 and recites: “… wherein the reverse transcriptase, including M-MLV reverse transcriptase or a functional variant thereof …”. This is confusing because claim 3 already requires an M-MLV reverse transcriptase or a functional variant thereof, so the recitation of “the reverse transcriptase” is referring to an “M-MLV reverse transcriptase or a functional variant thereof”. What does it mean for said transcriptase to be “including” an “M-MLV reverse transcriptase or a functional variant thereof” when it already is one?
Claim 8 recites the limitation "the functional variant of the M-MLV reverse transcriptase of which the RNase H domain is deleted" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim because it depends from claim 6 which does not recite an “M-MLV reverse transcriptase of which the RNase H domain is deleted”.
Claim 8 is also indefinite for reciting “… and preferably, the function variant of the M-MLV reverse transcriptase of which the RNase H domain is deleted is fused at the N-terminus…”, and this makes it unclear if the N-terminal fusion is required or if it is just optional. Are “preferable” limitations required by the claim?
Inadequate Written Description
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 3, 6-8, and 14 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim.
The claims are broadly drawn to a system that utilizes any reverse transcriptase (claim 1) including a system using an “M-MLV reverse transcriptase or a functional variant thereof” (claim 3).
Applicant describes mutated versions of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) that have the following amino acid substitutions relative to SEQ ID NO: 3: F155Y, F155V, F156Y, D524N, and/or D200C (Spec 8, 18; Fig. 1). Applicant describes 7 functional variants of M-MLV RT as SEQ ID NOs: 9-15, which are non-elected inventions (Spec 9 and 18; Fig. 1). Applicant describes two M-MLV RT variants in which the RNase H domain has been deleted as SEQ ID NOs: 14 and 15 which are non-elected inventions (Spec 18; Fig. 1).
Applicant describes transformation of rice protoplasts with vectors expressing their constructs (Spec 18-19). Applicant describes using PCR to amplify DNA fragments from the transformed rice protoplasts and sending the PCR products for sequencing (Spec 20-21).
Applicant describes three of their constructs as having an improved editing efficiency relative to the original system in which M-MLV RT was not modified; and those three constructs are: a construct in which the RNase H domain has been deleted and two constructs in which a nuclear capsid (NC) protein has been fused to the M-MLV RT; one to the N-terminus and the other to the C-terminus (Spec 22, Example 1, Figs. 1 and 2). The construct with the RNase H domain deleted is referred to as PPE-DRNase H and encodes the protein of SEQ ID NO: 14 (Spec 18, Material and method; Fig. 1). Applicant refers to the construct in which the NC protein is fused to the N-terminus of the M-MLV RT as SEQ ID NO: 19, elected invention (Spec 18).
Applicant describes testing the three successful constructs to see if they were improved in efficiency for targeting specific genomic loci, and the construct encoding SEQ ID NO: 19 (NC fused to the N-terminus of M-MLV RT), consistently provided the best efficiency for editing the specific loci (Fig. 3).
Applicant describes combining the N-terminal NC fusion with the deleted RNase H domain to result in a construct they refer to as ePPE and demonstrated a further improvement to their editing efficiency (Spec 23, Fig 4).
Applicant describes Western Blot analysis showing an increased accumulation of the M-MLV RT protein variants relative to the unmodified M-MLV RT (Spec 23, Fig. 5).
Applicant describes using ePPE to insert fragments having 18-34 bp at 18 different sites in the genome and using ePPE to delete fragments having 15-90 bp at 18 different sites (Spec 23-24, Fig. 6).
Applicant describes efficiency of targeting mutations in stably transformed rice calli at four specific loci as being 31.5% higher for ePPE compared with the unmodified control (PPE) (Spec 24, Fig. 8).
Applicant describes higher efficiency of introducing a specific point mutation in rice; 11.3% vs. 0.6% (Spec 24, Fig. 9).
Applicant describes improved efficiency of ePE for editing three targets in a pig cell (Spec 24, Fig. 10).
Applicant describes no significant increase in off-target editing compared to PPE (Spec 24-25).
Applicant describes an increase in targeted editing at sites in which the PAM sequences lacked a C or an A (Spec 25).
Applicant does not describe any prime editing systems using any reverse transcriptase other than the systems expressing SEQ ID NOs: 9-15 (not elected) and SEQ ID NO: 19 (examined). Applicant does not describe any M-MLV RT functional variants other than SEQ ID NOs: 9-15 and 19.
Given the unlimited number of RT variants encompassed by the instant claims, and given the fact the Applicant only reduces to practice 13 constructs encoding variants 3 of which do not appear to successfully edit the genome (Figs 1 and 2), Applicant has not provided an adequate written description to encompass the myriad variants encompassed by the claims.
NOTE: claim 14 would be adequately described if the full-length protein of SEQ ID NO: 19 were required, however the recitation of “an amino acid sequence of SEQ ID NO: 19” (emphasis added) renders the claim inclusive of small fragments of SEQ ID NO: 19, even as small as di-peptides.
Insufficient Scope of Enablement
Claims 3, 6-8, and 14 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a system for genome editing that includes a construct expressing M-MLV RT with a nuclear capsid protein fused to the N-terminus, does not reasonably provide enablement for a system for genome editing that includes a construct expressing M-MLV RT with a protease fused to either the N-terminus or the C-terminus. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
The claims are broadly drawn to a system that utilizes any reverse transcriptase (claim 1) including a system using an “M-MLV reverse transcriptase or a functional variant thereof” (claim 3) and including a system using an M-MLV RT fused with a protease or NC or integrase (claim 6).
Applicant teaches transformation of rice protoplasts with vectors expressing their constructs (Spec 18-19). Applicant describes using PCR to amplify DNA fragments from the transformed rice protoplasts and sending the PCR products for sequencing (Spec 20-21).
Applicant discloses that three of their constructs had an improved editing efficiency relative to the original system in which M-MLV RT was not modified; and those three constructs are: a construct in which the RNase H domain has been deleted and two constructs in which a nuclear capsid (NC) protein has been fused to the M-MLV RT; one to the N-terminus and the other to the C-terminus (Spec 22, Example 1, Figs. 1 and 2). Applicant refers to the construct in which the NC protein is fused to the N-terminus of the M-MLV RT as SEQ ID NO: 19, elected invention (Spec 18).
Applicant discloses the failure to successfully edit the genome for constructs expressing M-MLV RT fused to a protease at either the N-terminus or the C-terminus and a failure for a construct in which the RNase H domain and “connection” have been deleted (Fig. 2).
Given Applicant’s own data demonstrating that fusions with a protease were not able to successfully edit the genome in rice protoplasts, and given the breadth of the claims which include any versions of any reverse transcriptase (claim 1) or any functional variant of M-MLV RT (claim 3), the claims are not enabled through the broad scope encompassed by the claims.
NOTE: claim 14 would be enabled if the full-length protein of SEQ ID NO: 19 were required, however the recitation of “an amino acid sequence of SEQ ID NO: 19” (emphasis added) render the claim inclusive of small fragments of SEQ ID NO: 19, even as small as di-peptides.
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.
Anticipation by Gao
Claim(s) 1, 3, and 14 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by each of Gao et al. WO/2021/082830 (publication of PCT/CN2020/117736, published on May 6, 2021) and under 35 U.S.C. 102(a)(2) as being anticipated by Gao et al. (US Pre-Grant Publication US 2023/0075587; published on Mar. 9, 2023, for US Application No. 17/773,426 with priority to Nov. 1, 2019). The US Publication is for the 371 National Stage entry of the PCT application published in the WIPO document. The Examiner will refer to the US Publication page numbers in this Office Action because it is the English translation of the WIPO document.
The claims are directed to a prime editing system comprising or expressing a fusion protein comprising a CRISPR nickase and a reverse transcriptase (RT) and/or a pegRNA, wherein the pegRNA comprises a guide sequence, a scaffold sequence, an RT template sequence, and a primer binding site (PBS) sequence, wherein the pegRNA is capable of forming a complex with the fusion protein resulting in a nick being formed in the targeted sequence (claim 1), including wherein the RT is M-MLV RT or a functional variant thereof (claim 3), and including wherein the fusion protein contains “an” amino acid sequence of SEQ ID NO: 19 (claim 14). As discussed, above, in claim interpretation, claim 14 requiring “an amino acid sequence of SEQ ID NO: 19” only requires a di-peptide fragment of SEQ ID NO: 19.
Gao teaches targeted modification of a plant genome sequence using a nuclease-reverse transcriptase fusion protein guided by a guide RNA (Gao abstract). Gao claims a genome editing system comprising a fusion protein and/or an expression construct encoding the fusion protein, wherein the fusion protein comprises a CRISPR nickase and a reverse transcriptase and/or at least one pegRNA, wherein the pegRNA comprises a guide sequence, a scaffold sequence, a reverse transcription template sequence and a primer binding site, wherein the pegRNA can form a complex with the fusion protein to target a sequence in the genome and cause a nick in the target sequence (Gao 17, claim 1).
Gao teaches this system using an M-MLV reverse transcriptase, including using and “enhanced” M-MLV reverse transcriptase which is a “variant” (Gao 17, claim 3). M-MLV RT inherently comprises “an” amino acid sequence of SEQ ID NO: 19.
The applied reference has a common inventor and assignee with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
For the rejection under 35 USC 102(a)(1), Applicant would be required to provide a certified translation of their priority document to potentially be eligible for an exception under 35 U.S.C. 102(b)(1)(A).
Anticipation by Anzalone
Claim(s) 1, 3, and 14 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Anzalone et al. (Nature (2019) Vol. 576; pp. 149-157).
The claims are directed to a prime editing system comprising or expressing a fusion protein comprising a CRISPR nickase and a reverse transcriptase (RT) and/or a pegRNA, wherein the pegRNA comprises a guide sequence, a scaffold sequence, an RT template sequence, and a primer binding site (PBS) sequence, wherein the pegRNA is capable of forming a complex with the fusion protein resulting in a nick being formed in the targeted sequence (claim 1), including wherein the RT is M-MLV RT or a functional variant thereof (claim 3), and including wherein the fusion protein contains “an” amino acid sequence of SEQ ID NO: 19 (claim 14). As discussed, above, in claim interpretation, claim 14 requiring “an amino acid sequence of SEQ ID NO: 19” only requires a di-peptide fragment of SEQ ID NO: 19.
Anzalone teaches a prime editing complex with an RNA-guided DNA-nicking domain, such as Cas9 nickase, fused to an RT domain and complexed with a pegRNA resulting in DNA edits at specific positions (Anzalone 150, Figure 1 legend). The peg RNA comprises a guide RNA, a scaffold, an RT template sequence and primer binding site sequence (Id. Extended data, Fig. 3a legend). Anzalone teaches successful editing which necessarily involved nicking the target genomic site (Id. extended data Fig. 3). Anzalone teaches using the M-MLV RT (Id.). M-MLV RT inherently comprises “an” amino acid sequence of SEQ ID NO: 19.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 6-8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Anzalone et al. (Nature (2019) Vol. 576; pp. 149-157) in view of Lyu et al. (Life (2020) Vol. 10 pp. 1-16).
The claims are directed to a prime editing system comprising or expressing a fusion protein comprising a CRISPR nickase and a reverse transcriptase (RT) and/or a pegRNA, wherein the pegRNA comprises a guide sequence, a scaffold sequence, an RT template sequence, and a primer binding site (PBS) sequence, wherein the pegRNA is capable of forming a complex with the fusion protein resulting in a nick being formed in the targeted sequence, wherein the RT is M-MLV RT or a functional variant thereof, and wherein the reverse transcriptase is fused at either the N-terminus or the C-terminus with a nucleocapsid protein (NC), a protease (PR), or an integrase (IN) directly or via a linker in the fusion protein, including wherein the NC comprises SEQ ID NO:6, the PR comprises SEQ ID NO: 7 or the IN comprises SEQ ID NO: 8. As discussed, above, in claim interpretation, claim 7 requiring “an amino acid sequence of SEQ ID NO: 6” only requires a di-peptide fragment of SEQ ID NO: 6; the sequences for the PR and IN only require a di-peptide fragment as well.
Anzalone teaches a prime editing complex with an RNA-guided DNA-nicking domain, such as Cas9 nickase, fused to an RT domain and complexed with a pegRNA resulting in DNA edits at specific positions (Anzalone 150, Figure 1 legend). The peg RNA comprises a guide RNA, a scaffold, an RT template sequence and primer binding site sequence (Id. Extended data, Fig. 3a legend). Anzalone teaches successful editing which necessarily involved nicking the target genomic site (Id. extended data Fig. 3). Anzalone teaches using the M-MLV RT (Id.). M-MLV RT inherently comprises “an” amino acid sequence of SEQ ID NO: 19.
Anzalone does not teach an RT fused to an NC.
Lyu teaches the delivery of a nuclease, including CRISPR/Cas, using a virus-like particle (Lyu, title and abstract). Lyu teaches the strategy of fusing the nuclease to the N-terminus of Gag protein to package the nuclease in a virus-like particle (VLP) (Lyu 7). Lyu teaches that the Gag protein comprises an NC (Id. 4-5 and 6, Fig. 3). The NC taught by Lyu would have inherently comprised a di-peptide fragment of SEQ ID NO: 6.
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to modify the prime editing system taught by Anzalone by fusing it to a Gag protein for packaging into a VLP as taught by Lyu. Lyu provides examples of Cas9 fused indirectly at its C-terminus to NC (Lyu 6, Figure 3, Hotta’s or Manjunath’s group) and examples of Cas9 fused at its N-terminus to NC (Id., Ricel’s or Indik’s group). One would be motivated to package the prime editing system into a VLP because Lyu teaches several advantages for VLP-delivery of Cas9 including no need to concentrate the VLPs, ease of production, significantly less product needed, and greater specificity (Id. 9).
Summary
No claim is allowed.
Examiner’s Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHY KINGDON whose telephone number is (571)272-8784. The examiner can normally be reached M-F 9:00 - 5:30 EST.
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CATHY KINGDON
Primary Examiner
Art Unit 1663
/CATHY KINGDON/ Primary Examiner, Art Unit 1663
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