DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application Status and Election
Applicant’s amendments filed December 5, 2025 amending claims 1 and 11, and canceling claims 16-20 are acknowledged. Applicant's election without traverse of Group 1 in the reply filed on December 5, 2025 is acknowledged.
Claims 1-15 are pending and under examination.
Drawings
The drawings are objected to because:
First, the figures are referred to as “Figure” in the drawings. MPEP §608.02.V states that according to 37 C.F.R. 1.84(u)(1) “View numbers must be preceded by the abbreviation "FIG.".
Second the numbers and letters of FIGs 3, 5, 6 and 8A-C are not sufficient to provide satisfactory reproduction characteristics. 37 CFR 1.84(l) states that “all drawings must be made by a process which will give them satisfactory reproduction characteristics. Every line, number, and letter must be durable, clean, black (except for color drawings), sufficiently dense and dark, and uniformly thick and well-defined.” In the instant case, the text in the above listed FIGs is light grey or otherwise not sufficiently dense and dark to permit satisfactory reproduction characteristics; and/or are is very small and of poor resolution.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Interpretation
Claim 1 recites “a library of modular nucleic acid sequences”. The Specification does not define library. The broadest reasonable interpretation of “library” is two or more modular nucleic acid sequences. Additionally, by virtue of the inherent nature of nucleic acids to be ligated and cleaved from each other, any nucleic acid sequence is interpreted to be “modular”.
Claim 1 also recites “site directed nuclease modules each comprising a nucleic acid sequence encoding a different site-directed nuclease”. In the art, nuclease typically means having the ability to cleave nucleic acids, which requires the enzymatic function of cleaving a phosphodiester bond of the sugar-phosphate backbone. However, the Specification defines a “site-directed nuclease” as one that “binds one or more nucleotide sequence (e.g. DNA or RNA) and exerts (or not) one of multiple modifications on them” ([0032]). Therefore a “site-directed nuclease” does not necessarily have nuclease activity. The specification lists “an enzyme containing zinc-fingers which binds DNA at specific sites” as a nuclease ([0032]). Therefore, the genus of “site-directed nucleases” is interpreted to include a zinc-finger (ZF) DNA binding domain, a transcription activator like effector (TALE) DNA binding domain, and nuclease active and deficient Cas effectors such as Cas9, Cas12a, nCas9, dCas9, and dCas12a.
Claim objections
Claim 13 is objected to because of the following informalities:
The last two lines in claim 13 recites “wherein each nucleic acid assembly encodes the site-directed nuclease, the nucleotide-modifying enzyme”, which is grammatically incorrect. There needs to be a conjunction joining the two nouns.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 5 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 5, option (i) recites the method of claim 1, wherein step (b) comprises: (i) constructing a combinatorial library of nucleic acid assemblies in vitro. Claim 1, step (b) already requires the assembly to be in vitro. Claim 1 step (b) also recites constructing two ore more nucleic acid assemblies, wherein each assembly is constructed by joining a site-directed nuclease module with a nucleotide modifying enzyme module. Because a “library” is interpreted as two or more entities and the library of claim 1 requires different nuclease modules and different nucleotide-modifying enzymes in each assembly, the library of claim 1 is already a “combinatorial library”. Therefore, claim 5, option (i) does not appear to further limit the subject matter of claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 112(b)
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.
Claim 6 is 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.
Claim 6 recites the limitation "one of the additional modules" in lines 3-4. There is insufficient antecedent basis for this limitation in the claim. No “additional modules” are recited in claims 1 or 5, from which claim 6 depends, or previously in claim 6. Therefore, it is unclear what “the additional modules” is referring to.
Claim Rejections - 35 USC § 102 - Yang
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-8 and 12-15 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Yang (Yang et al., Nature Communications (2016), 7: 13330, and Supplemental Material).
Regarding claim 1, step (a), Yang teaches a general design for targeted deaminases comprising a zinc finger (ZF) or a transcription activator like effector (TALE) DNA binding domain (i.e., two different site-directed nucleases) fused to an AID or APOBEC deaminase domain (i.e., two different nucleic-modifying enzymes) (Fig 1a; page 2, ¶5-7). Yang teaches linking the nucleic acids encoding the ZF or TALE together with the nucleic acids encoding the different deaminase domains (Supp Fig 1). Regarding step (b), Yang teaches constructing two assemblies in vitro by joining the nucleic acid encoding ZF or TALE with the nucleic acid encoding AID or APOBEC such that they are operably linked (Supp Fig 1). Regarding steps (c) and (d), Yang teaches expressing the ZF/TALE-deaminase fusions in bacterial cells and detecting the deamination frequency (i.e., the nucleotide-modifying activity for each fusion polypeptide) (Fig 1c-f).
Regarding claims 2 and 12, Yang teaches analyzing the deamination frequency of each fusion polypeptide to determine which had the highest frequency (i.e., which is the optimal assembly) (Fig 1c-f; page 2, ¶5-7).
Regarding claims 3 and 4, option (ii), Yang teaches the nucleotide-modifying enzymes are deaminases that are APOBEC and AID enzymes (Fig 1).
Regarding claims 5-7, Yang teaches constructing two vectors each comprising either the ZF or the TALE modules (i.e., constructing two or more destination vectors comprising the nuclease module) by mixing together molecules each comprising either the nuclease module of the deaminase module (Supp Fig 1). Yang teaches the ZF and TALE nucleic acids are in plasmids (i.e., a vector). Yang teaches the deaminase is encoding by a double-stranded piece of DNA flanked by restriction sites (i.e., a vector) (Supp Fig 1).
Regarding claim 8, Yang teaches the vectors comprising the ZF and TALE modules comprises a pTrc promoter driving the ZF/TALE expression (i.e., a promoter operably linked for expression of the fusion polypeptides) (Supp Fig 1).
Regarding claim 13-15, Yang’s teachings regarding a library of fusion proteins comprising (i) either ZF or TALE, and (ii) either APOBEC or AID is recited above as for claims 1 and 3-4 (Fig 1).
Claim Rejections - 35 USC § 102 - Li
Claims 1-8 and 12-15, are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Li (Li et al., Nature Biotechnology (2018), 36: 324-327, and Supplemental Material).
Regarding claim 1, step (a), Li teaches dAsCpf1, dLbCpf1, and nCas9 (i.e., at least two different site-directed nucleases) fused either BE, BE2 BE3, BE-YE or eBE-YE (i.e., at least two different nucleic-modifying enzymes) (Figs 1-2; Supp Fig 1a). Li teaches linking the nucleic acids encoding the dAsCpf1, dLbCpf1, or nCas9 together with the nucleic acids encoding the different deaminase domains (Methods, column 1; Supp Fig 1). Regarding step (b), Li teaches constructing two assemblies in vitro by joining the nucleic acid encoding dAsCpf1 or dLbCpf1 with the nucleic acid encoding the APOBEC deaminase or modified forms of APOBEC such that they are operably linked (Methods, column 1; Supp Fig 1). Regarding steps (c) and (d), Li teaches expressing the dAsCpf1, dLbCpf1 or nCas9 deaminase fusions in mammalian cultured cells and detecting the deamination frequency (i.e., the nucleotide-modifying activity for each fusion polypeptide) (Methods, column 2, ¶4-6; Figs 1-2).
Regarding claims 2 and 12, Li teaches analyzing the deamination frequency of each fusion polypeptide to determine which had the highest on-target frequency (i.e., determining the optimal assembly) (Figs 1-2; Methods, page 1, last ¶, and page 2).
Regarding claims 3 and 4, option (i), Li teaches the site-directed nucleases are dLbCpf1 (i.e., LbCas12a), dAsCpf1 (i.e., AsCas12a), and the nCas9 as reported in Komor et al., (i.e., SpCas9) (page 324, ¶3-4). Regarding option (ii), Li teaches the nucleotide-modifying enzymes are deaminases that are APOBEC enzymes (Fig 1).
Regarding claim 5-7, Li teaches destination vectors comprising either dAsCpf1 or dLbCpf1 were constructed by mixing PCR fragments encoding either dAsCpf1 or dLbCpf1 with an NLS-comprising linearized vector (i.e., a molecule comprising an additional module) to produce expression constructs for NLS-fused dAsCpf1 or dLbCpf1 (Methods, ¶2).
Regarding claim 8, Li teaches the pST1374 vectors also comprises the pCMV promoter for expression of the NLS-fused dAsCpf1 or dLbCpf1 (Methods, ¶2).
Regarding claim 13-15, Li’s teachings regarding a library of fusion proteins comprising (i) either nCas9, AsCpf1 or LbCpf1, and (ii) either BE, eBE or BE-YEE is recited above as for claims 1 and 3-4 (Figs 1-2).
Claim Rejections - 35 USC § 103 – Yang in view of Fonseca
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.
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 9 is rejected under 35 U.S.C. 103 as being unpatentable over Yang (Yang et al., Nature Communications (2016), 7: 13330, and Supplemental Material) as applied to claims 1-8 and 12-15 above, and further in view of and Fonseca (Fonseca et al., ACS Synthetic Biology (2019), 8: 2593-2606).
The teachings of Yang are recited above in paragraphs 22-27 as for claims 1-8 and 12-15, and are incorporated here. Briefly, Yang teaches assaying combinations of ZF or TALE DNA binding domain (i.e., nucleases) with different deaminases to determine optimal nuclease-deaminase combinations for base editing. Yang also teaches a deamination assay using a genomically-integrated EF1a promoter (i.e., a third promoter) operably linked to a ZF-binding site (i.e., a target site) which is linked to a green fluorescent protein (GFP) coding sequence (i.e., a reporter sequence) (Fig 4a). Yang teaches the reporter expression construct comprising a modified GFP coding sequence which has a mutated start codon in its non-edited form which prevents expression of GFP (Fig 4d). Yang teaches the GFP reporter expression construct cell line was generated by lentiviral transduction/integration and isolated using FACS screening (page 6, ¶3).
Yang does not teach including the target-reporter expression cassette in the plasmids comprising the coding sequence for either the nucleases or deaminase modules.
Fonseca teaches a Golden-Gate cloning-based cloning toolkit for cloning modular elements into large DNA vectors and implementation in mammalian models (Abstract). Fonseca teaches a destination vector comprising a mAzami green coding sequence operably linked to either the pUAS or pTRE reporter (i.e., a target-reporter expression cassette comprising a target sequence and a fluorescent reporter sequence operably linked to a third reporter) (Fig 5). Fonseca teaches the destination vector also comprises the sequence for either dSaCas9 or dSpCas9 (i.e., two or more nucleases) operably linked to KRAB or VPR, which are transcriptional regulator domains that are recruited to the target DNA by Cas9 (Fig 5). Thus, Fonseca teaches the nuclease-effector coding sequence in the same vector as the reporter construct.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have additionally included Yang’s GFP reporter expression construct in the vectors encoding the ZF/TALE modules. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that the GFP reporter construct could be included in the vectors because Fonseca teaches that reporter constructs can be included on the same vector as the fusion protein being evaluated. One would have been motivated to do so to test the deamination assays in different cell types since Yang’s integrated reporter cannot be easily transferred to another cell type. By including the reporter in the same plasmid as the nuclease-deaminase fusion, the skilled artisan could eliminate a step needing to integrate the reporter expression construct that was used in Yang.
Claim Rejections - 35 USC § 103 – Li in view of Fonseca and Jiang
Claims 9-11 is rejected under 35 U.S.C. 103 as being unpatentable over Yang (Yang et al., Nature Communications (2016), 7: 13330, and Supplemental Material) as applied to claims 1-8 and 12-15 above, and further in view of and Fonseca (Fonseca et al., ACS Synthetic Biology (2019), 8: 2593-2606) and Jiang (Jiang et al., Cell Research (2018), 28: 855-861).
The teachings of Li are recited above as for claims 1-8 and 12-15 and are incorporated here. Briefly, Li teaches assaying combinations of Cas9 or Cas12a nucleases with different deaminases to determine optimal nuclease-deaminase combinations for base editing. Li teaches targeting endogenous loci in HEK293 cells, such as DNMT1 and EMX1, and assaying base editing in the endogenous genes.
Li does not teach including a target-reporter expression cassette in the plasmids comprising the coding sequence for either the nucleases or deaminase modules.
Fonseca teaches a Golden-Gate cloning-based cloning toolkit for cloning modular elements into large DNA vectors and implementation in mammalian models (Abstract). Fonseca teaches a destination vector comprising a mAzami green coding sequence operably linked to either the pUAS or pTRE reporter (i.e., a target-reporter expression cassette comprising a target sequence and a fluorescent reporter sequence operably linked to a third reporter) (Fig 5). Fonseca teaches the destination vector also comprises the sequence for either dSaCas9 or dSpCas9 (i.e., two or more nucleases) operably linked to KRAB or VPR, which are transcriptional regulator domains that are recruited to the target DNA by Cas9 (Fig 5). Thus, Fonseca teaches the nuclease-effector coding sequence in the same vector as the reporter construct.
Jiang teaches engineering cytidine base editor systems comprising nCas9 variants and an APOBEC deaminase domain (Abstract). Jiang teaches assaying deamination of the base editor systems using the GFP-iSTOP assay (page 858, ¶1; Fig 4). Jiang teaches the GFP-iSTOP cassette comprises a sequence with comprising TGG codons fused to the GFP coding sequence (i.e., the target sequence and the reporter sequence are operably linked to express as a single reporter polypeptide) (Fig 4b). Jiang teaches designing guide RNAs to target the target sequence that is fused to GFP (Fig 4b). Jiang teaches the GFP-iSTOP assay involves converting a C into T at four potential nucleotides that would produce a stop codon from CAG, CGA, CAA and TGG (i.e., editing one or more of the target nucleotides results in the formation of an edited sequence having one or more stop codons (page 858, ¶1; Fig 4b). Jiang teaches when the target sequence is edited, GFP expression is knocked down (i.e., the editing results in non-expression of the reporter polypeptide) (Fig 4c). Jiang teaches (i) identifying the modified site in the edited targeted sequence by the two base editors BE3 and BE3-PLUS (Fig 4d). Although Jiang is silent about the specific promoter driving expression of the GFP-iSTOP cassette, the GFP-iSTOP cassette must have inherently included a promoter operably linked to it (i.e., a third promoter) because Jiang teaches that GFP is expressed (Fig 4c).
Regarding claims 9-11, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have additionally included a fluorescent reporter expression construct, such as Jiang’s GFP-iSTOP reporter, in Li’s vectors encoding the nuclease modules. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that the GFP-iSTOP reporter could be included in the vectors because Fonseca teaches that reporter constructs can be included on the same vector as the DNA-binding protein fusion being evaluated. One would have been motivated to do so to test the deamination assays in different cell types since Li’s assay only allows targeting human genes. By including the reporter in the same plasmid as the nuclease-deaminase fusion, the skilled artisan could standardize the assay to assess deamination activity of the different nuclease-deaminase combinations in different cell types. The skilled artisan would have been specifically motivated to use the GFP-iSTOP reporter since Jiang demonstrates its usefulness in assaying deamination efficiency when screening for base editors with high on-target activity and to identify the editing window of the deaminase.
Conclusion
No claims are allowable.
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/CATHERINE KONOPKA/Examiner, Art Unit 1635