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Application Status and Withdrawn Rejections
Applicant’s amendments filed May15, 2026 amending claims 1, 3, 5-6 and 13-15 are acknowledged. Claims 1-15 are pending and under examination.
The amendments to the claim requiring at least three site-directed nuclease modules, at least three nucleotide-modifying enzyme modules, and specific cloning steps to construct the combinatorial assemblies overcomes the §102 and §103 rejections of record.
Any other rejection or objection not reiterated herein has been overcome by amendment. Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Drawings
The drawings submitted May 15, 2026 are objected to because:
The numbers and letters of FIGs 1-7 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: specifically the light gray text in the boxes of FIG. 1, the restriction enzyme label in FIG. 3, all of the text in FIG. 4, the labels in the legend and on the x- or y-axis in FIGs. 5 and 6, and the labels next to the enzyme structure in FIG 7. Additionally, the text over shading in FIGs 2, 4 and 7 is not sufficiently dark compared to the background shading to be legible.
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 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.
Claims 1-15 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. This is a new rejection necessitated by amendment.
Claim 1 is indefinite for two reasons:
First, claim 1 recites “a method for obtaining assemblies of site-directed nucleases (SDNs) and nucleotide-modifying enzymes (NMEs), the method comprising (a) providing a library of modular nucleic acid sequences configured for combinatorial assembly of different nuclease-deaminase combinations, the library comprising… (ii) three or more nucleotide-modifying enzyme modules…”. Deaminases are one just type of nucleotide-modifying enzymes. Including both the broader genus of “nucleotide-modifying enzymes” with the narrower genus “deaminase” genus in the same claim is confusing because it is not clear if the NMEs must be deaminases or if they can be any NME like methylases or demethylases.
Claim 1 also recites “comprising: (a) providing a library of modular nucleic acid sequences…, the library comprising (i) three or more SDN modules… (ii) three or more NME modules, (b) combinatorial constructing three or more nucleic assemblies…, wherein at least two nucleic assemblies comprise different combination of SDNs and MNEs…, wherein three or more nucleic acid assemblies are constructed in parallel in a single reaction; and expressing the three or more nucleic acid assemblies constructed in step (b)… Claim 1 is confusing because it is not clear what is the minimum number of different combinations of SDNs and MNEs required in the final library of assemblies. Step (b) only requires two assemblies to have different combinations, which would produce only two different fusion combinations. But step (c) requires at least three nucleic acid assemblies, which is three different combinations. Additionally, step (a) requires at least three different SDNs and three different NMEs. It’s unclear why three of each is needed if only two or three different combinations are ultimately produced. Using three of each would result in a combinatorial library of nine different combinations. As such, it is not clear how many different combinations of SDNs and NMEs assemblies are produced in the library assembly step.
Claims 2-12 are rejected for depending from claim 1 and not remedying either the first and/or second source of indefiniteness.
Claim 13 is indefinite for two reasons.
First, claim 13 recites “a library of nucleic acid assemblies of site-directed nucleases (SDNs) and nucleotide-modifying enzymes (NMEs) configured for combinatorial assembly of different nuclease-deaminase combinations, comprising… (ii) three or more nucleotide-modifying enzyme modules…”. Deaminases are one just type of nucleotide-modifying enzymes. Including both the broader genus of “nucleotide-modifying enzymes” with the narrower genus “deaminase” genus in the same claim is confusing because it is not clear if the NMEs must be deaminases or if they can be any NME like methylases or demethylases.
Second, claim 13 is directed to a library of assemblies of the SDNs and the NMEs, and thus the nucleic acids encoding the SDNs and NMEs must already be assembled in an open reading frame. However, claim 13 also recites that the SDN modules and NME modules have 5’ and 3’ overhangs. It is confusing how the modules that are already assembled can have 5’ and 3’ overhangs since in the assemblies the overhangs would have annealed and been ligated together.
Claims 14-15 are rejected for depending from claim 13 and not remedying either the first and/or second source of indefiniteness.
Claim 6 is also indefinite because the required elements in the destination vector is not clear. Claim 6 recites “mixing two or more nucleic acid molecules each comprising one of the SDN modules, one of the NME modules, one or more linker sequences, nuclear localization signal sequences, or UGI sequences, a protein coding sequence, a regulatory sequence, or any combination thereof…” The only conjunction used in claim 6 is “or” and it is used twice. Claim 6 depends from claim 1 which requires an assembly of at least the SDN and the NME, thus it appears that at least a nucleic acid molecule comprising an SDN module and a nucleic acid molecule comprising an NME module is required. But it is not clear if any additional element is required. Also, it is not clear what the relationship is between a “nucleic acid molecule” and the “nucleic acid assembly”.
Claim 7 is indefinite for depending from claim 6 and not remedying the indefiniteness. It is not also not clear what the relationship is between the nucleic acid “assemblies”, “molecules”, “donor vectors” or “receiver vectors”. If the modules are comprised within a “vector” it is not clear how they could have 5’ and/or 3’ overhangs and be assembled.
If Applicant intends for at least one additional module besides the SDE and NME to be present in the destination vector, the following claim language is suggested: Claim 6: The method of claim 5, wherein constructing the two or more destination vectors comprises mixing (1) a nucleic acid molecule comprising one of the nucleic acid assemblies comprising a site-directed nuclease module and a nucleotide-modifying enzyme module with (2) a nucleic acid molecule comprising one or more linker sequences, nuclear localization signal sequences, UGI sequences, a protein coding sequence, a regulatory sequence, or any combination thereof.
Claim Interpretation
Claims 1 and 13 are interpreted as at least three different combinations of SDNs and generic NMEs are produced in the assembly library since at least three different assemblies are needed.
Because claim 13 requires the library to be a library of assemblies, the 5’ overhang and 3’ overhang limitations between the SDE and the NME modules will be interpreted as not present.
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”.
Also, as explained in the previous office action (paragraph 9), a site-directed nuclease is interpreted as a DNA-binding domain and includes 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 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 13-15 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, of record). This is a new rejection necessitated by amendment.
As noted above in paragraph 22, because claims 13-15 are directed to the library of assemblies comprising the ligated/connected SDE and NME modules, the limitations reciting 5’ and/or 3’ overhangs are interpreted as not limiting the structure of the assemblies in the library.
Regarding claims 13-15, Yang teaches targeted deaminases comprising a zinc finger (ZF) or one of 5 different transcription activator like effector (TALE) DNA binding domains C1 through C5 (i.e., at least three different site-directed nucleases) fused to AID, APOBC1, ABOBEC3F and/or APOBEC3G deaminase domain (i.e., at least three different nucleic-modifying enzymes) (Fig 1a, Fig 2; page 2, ¶5-7). Yang teaches the following fusion proteins: ZF-APOBC1, ZF-APOBEC3F, ZF-APOBEC3G, ZF-AID, TALE-C1-AID, TALE-C1-AID, TALE-C2-AID, TALE-C3-AID, TALE-C4-AID and TALE-C5-AID (i.e., 6 different site-directed nuclease domains, 4 different deaminase domain, and 10 different combinations) (Figs 1-2).
Claim Rejections - 35 USC § 102 - Li
Claims 13-15 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, of record). This is a new rejection necessitated by amendment.
Regarding claim 13-15, Li teaches dAsCpf1, dLbCpf1, and nCas9 (i.e., at least three different site-directed nucleases) fused either BE, BE2, BE3 (i.e., rAPOBEC1), BE-YE or YEE (i.e., at least two different nucleic-modifying enzymes) (Figs 1-2; Supp Fig 1a). Li teaches the following fusion proteins: dAsCpf1-APOBEC1, dLbCpf1-APOBEC1, nCas9-APOBEC1, dLbCpf1-YE, dLbCpf1-YEE (i.e., 3 different site-directed nuclease domains, 3 different deaminase domain, and 5 different combinations) (page 324, ¶3; Figs 1-2).
Response to Arguments - §102
Applicant argues that Yang and Li do not teach assembly of the Cas/ZF/TALE nuclease modules with the deaminase modules wherein the nuclease modules each have the same overhangs and which were compatible with overhangs that are on the modules encoding the deaminase domains (Remarks 11-13). This argument has been fully considered and is persuasive as it pertains to the methods of assembly. However, claims 13-15 are directed to a “library of nucleic acid assemblies” such that the nuclease modules and deaminase modules are already assembled and the nucleic acid modules would no longer have 5’ and/or 3’ overhangs. The plasmids encoding the nuclease-deaminase fusion combinations of Yang and Li read on the claimed library of nucleic acid assemblies as discussed above.
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 1-8 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Yang (Yang et al., Nature Communications (2016), 7: 13330, and Supplemental Material, of record) in view of Fonseca (Fonseca et al., ACS Synthetic Biology (2019), 8: 2593-2606, of record). This is a new rejection necessitated by amendment.
Regarding claim 1, step (a), Yang teaches a general design for targeted deaminases comprising a zinc finger (ZF) or one of 5 different transcription activator like effector (TALE) DNA binding domain (i.e., at least three different site-directed nucleases, SDNs) fused to AID, APOBC1, ABOBEC3F or APOBEC3G deaminase domain (i.e., at least three different nucleic-modifying enzymes, NMEs) (Fig 1a, Fig 2; 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 using restriction enzyme digestion and ligation (i.e., having common 5’ and/or 3’ overhang sequences) (Supp Fig 1). Regarding steps (c) and (d), Yang teaches expressing at least three different ZF/TALE-deaminase fusion combinations in bacterial cells and detecting the deamination frequency (i.e., the nucleotide-modifying activity for each fusion polypeptide) (Fig 1c-f, Fig 2a-d).
Yang does not teach each NME module and each SDN module comprises the same 5’ and 3’ overhang sequences. Yang does not teach assembling the nuclease-deaminase fusion combinations in parallel in single reaction.
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 the bases of the modular toolkit is using a Golden Gate cloning reaction into a standard vector that utilizes type-IIS restriction enzymes BsaI and BsmBI that leaves four arbitrary base overhangs adjacent to the recognition site (Fig 1a; page 2595, ¶3). Fonseca teaches the steps for assembly are amplifying the modules via PCR to include BsaI and BsmBI sites on the 5’ and 3’ ends such that the overhang at the 3’ end of an upstream part is compatible with the overhand at the 5’ end of the downstream part (Fig 1a; page 2603, ¶4-11). Fonseca teaches the modular toolkit can also be used for assembling modules comprising the site-directed nucleases dSaCas9 and dSpCas9 fused to effector domains like KRAB and VPR (Fig 5). Fonseca teaches each of the nucleic acid modules were assembled in parallel in the same reaction (Figs 1 and 5, legend; page 2601, ¶3). Fonseca teaches the modular toolkit facilitates parallelization of large libraries of vector in one-step combinatorial reactions (i.e., the assemblies are constructed in parallel in a single reaction) (page 2597, ¶6). Fonseca teaches the modular toolkit assembly method is relevant for optimization of a large number of protein/Cas9 screens where variant representation is critical (page 2597, ¶6). Fonseca teaches the modular toolkit assembly method allows for the rapid and facile assembly of genetic circuits (page 2602, ¶7).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have assembled the ZF and TALE – deaminase libraries using the method of Fonseca such that each of the ZF and TALE coding sequences had the same ends which were compatible with the same ends for each of the deaminase coding sequences and assembled in a single reaction. The skilled artisan would have predicted that Fonseca’s assembly method could be used to assemble the library because 1) Fonseca demonstrates successful assembly of protein fusions, and 2) Yang teaches cloning the fusion proteins can involve compatible overhangs between the sequences encoding the DNA binding domain and the deaminase domain. The skilled artisan would have been motivated to use Fonseca’s parallel cloning methods because Fonseca describes it as rapid and facile for screening variants, such as the different deaminase and TALE variants in Yang, thereby eliminating the need for Yang’s laborious cloning strategy.
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, Fonseca teaches the modular toolkit assembly method requires each “part” to be first cloned into a parts library vector and then assembled with other “parts” to form a library of vectors comprising two or more parts (i.e., two or more destination vectors) (Fig 1a).
Claims 6 and 7 are indefinite for the reasons explained above in paragraphs 18 and 19. For the purposes of applying prior art and compact prosecution, the claims are interpreted as the destination vectors comprise an SDE module, an NME module, and an additional module that encodes one or more linker sequences, NLSs, UGIs, a protein coding sequence, or a regulatory sequence. “Vector” is interpreted as any piece of DNA.
Regarding claims 6-8, 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 testing the ZF/TALE-deaminase fusions in both bacterial cells with the T7 promoters or the pTrc promoter and in human cells using EF1a or CMV promoters (Fig 1, Supp Fig 1, Fig 4).
Fonseca teaches the modular assembly toolkit involves cloning each “part” into a plasmid (i.e., a donor vector), then release from the vector and combined via annealing and ligation of compatible ends to form a destination vector (Fig 1a). Fonseca teaches additional modules can comprise nucleic acid sequence comprising promoter sequences and polyadenylation sequences (i.e., regulatory sequences) (Figs 1 and 5).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have also included promoters and polyadenylation sequences as “parts” when constructing the destination vectors because Fonseca teaches promoters can be easily swapped out using the method. In Yang’s cloning method, each promoter needed to be cloned independently into the different ZF/TALE-deaminase fusion plasmids. By including the promoter as a modular “part” in the assembly method as taught by Fonseca, the skilled artisan could create the different combinatorial protein fusions with different promoters in a single reaction.
Claim Rejections - 35 USC § 103 – Li in view of Fonseca
Claims 1-8 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Li (Li et al., Nature Biotechnology (2018), 36: 324-327, and Supplemental Material, of record) in view of Fonseca (Fonseca et al., ACS Synthetic Biology (2019), 8: 2593-2606, of record). This is a new rejection necessitated by amendment.
Regarding claim 1, step (a), Li teaches dAsCpf1, dLbCpf1, and nCas9 (i.e., at least three different site-directed nucleases) fused either BE, BE2, BE3 (i.e., rAPOBEC1), YE or YEE (i.e., mutated rAPOBEC1) (i.e., at least three 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 five different combinations/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). Li teaches cloning the different dCas9/dCpf1-deaminase fusions by various separate PCR amplifications and separate ligations with compatible sticky ends to construct each individual fusion (Methods, ¶1-2, 5-8).
Li does not teach each NME module and each SDN module comprises the same 5’ and 3’ overhang sequences. Li does not teach assembling the nuclease-deaminase fusion combinations in parallel in single reaction.
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 the bases of the modular toolkit is using a Golden Gate cloning reaction into a standard vector that utilizes type-IIS restriction enzymes BsaI and BsmBI that leaves four arbitrary base overhangs adjacent to the recognition site (Fig 1a; page 2595, ¶3). Fonseca teaches the steps for assembly are amplifying the modules via PCR to include BsaI and BsmBI sites on the 5’ and 3’ ends such that the overhang at the 3’ end of an upstream part is compatible with the overhand at the 5’ end of the downstream part (Fig 1a; page 2603, ¶4-11). Fonseca teaches the modular toolkit can also be used for assembling modules comprising the site-directed nucleases dSaCas9 and dSpCas9 fused to effector domains like KRAB and VPR (Fig 5). Fonseca teaches each of the nucleic acid modules were assembled in parallel in the same reaction (Fig 1, Fig 5, legend; page 2601, ¶3). Fonseca teaches the modular toolkit facilitates parallelization of large libraries of vector in on-step combinatorial reactions (i.e., the assemblies are constructed in parallel in a single reaction) (page 2597, ¶6). Fonseca teaches the modular toolkit assembly method is relevant for optimization of a large number of protein/Cas9 screens where variant representation is critical (page 2597, ¶6). Fonseca teaches the modular toolkit assembly method allows for the rapid and facile assembly of genetic circuits (page 2602, ¶7).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have assembled the dCas9 or dCpf1 – deaminase libraries using the method of Fonseca such that each of the dCas9 and dCpf1 coding sequences had the same ends which were compatible with the same ends for each of the deaminase coding sequences and assembled in a single reaction. The skilled artisan would have predicted that Fonseca’s assembly method could be used to assemble the library because 1) Fonseca demonstrates successful assembly of protein fusions, and 2) Li teaches cloning the fusion proteins can involve compatible overhangs between the sequences encoding the DNA binding domain and the deaminase domain. The skilled artisan would have been motivated to use Fonseca’s parallel cloning methods because Fonseca describes it as rapid and facile for screening variants, such as the rAPOBEC1 variants and dCpf1 variants, thereby eliminating the need for Li’s laborious cloning strategy.
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, Fonseca teaches the modular toolkit assembly method requires each “part” to be first cloned into a parts library vector and then assembled with other “parts” to form a library of vectors comprising two or more parts (i.e., two or more destination vectors) (Fig 1a).
Claims 6 and 7 are indefinite for the reasons explained above in paragraphs 18 and 19. For the purposes of applying prior art and compact prosecution, the claims are interpreted as the destination vectors comprise an SDE module, an NME module, and an additional module that encodes one or more linker sequences, NLSs, UGIs, a protein coding sequence, or a regulatory sequence. “Vector” is interpreted as any piece of DNA.
Regarding claims 6-8, 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) and a plasmid comprising the pCMV promoter to produce expression constructs for NLS-fused dAsCpf1 or dLbCpf1 (Methods, ¶2). Li teaches the pST1374 vectors also comprises the pCMV promoter for expression of the NLS-fused dAsCpf1 or dLbCpf1 (Methods, ¶2).
Fonseca teaches the modular assembly toolkit involves cloning each “part” into a plasmid (i.e., a donor vector), then release from the vector and combined via annealing and ligation of compatible ends to form a destination vector (Fig 1a). Fonseca teaches additional modules can comprise nucleic acid sequence comprising promoter sequences and polyadenylation sequences (i.e., regulatory sequences) (Figs 1 and 5).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have also included the NLSs, promoters and/or polyadenylation sequences as “parts” when constructing the destination vectors because Fonseca teaches promoters can be easily swapped out using the method. In Li’s cloning method, each promoter, NLS, nuclease, and/or deaminase needed to be cloned and/or engineered independently into the different Cas-deaminase fusion plasmids. By including the promoter and NLS as a modular “part” in the assembly method as taught by Fonseca, the skilled artisan could create the different combinatorial protein fusions with different promoters and/or different NLSs in a single reaction.
Claim Rejections - 35 USC § 103 – Li in view of Fonseca and Jiang
Claims 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over Li (Li et al., Nature Biotechnology (2018), 36: 324-327, and Supplemental Material, of record) and Fonseca (Fonseca et al., ACS Synthetic Biology (2019), 8: 2593-2606, of record), as applied to claims 1-8 and 12 above, and further in view of Jiang (Jiang et al., Cell Research (2018), 28: 855-861, of record). This is a new rejection necessitated by amendment.
The teachings of Li and Fonseca are recited above and applied as for claims 1-8 and 12. Li also 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 also 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).
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-deaminase modules constructed using the method of Fonseca. 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.
Response to Arguments - §103
Applicant argues that Fonseca and Jiang do not remedy the deficiencies of Li or Yang because the cited teachings in Fonseca do not teach the parallel one-pot construction of three or more assemblies (Remarks 14-15). This argument has been fully considered but is moot as it pertains to the current rejection. As detailed above each of the primary references Yang and Li each teach 3 or more nuclease domains and 3 or more deaminase domains while Fonseca provides a high throughput, one-pot combinatorial cloning method using Golden Gate cloning (i.e., compatible 5’ and 3’ ends for directional cloning).
Conclusion
No claims are allowable.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571)272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
Prosecution Insights