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
This action is written in response to applicant’s correspondence received 11/13/2023. Claims 1-7, 13-22, and 28-30 are currently pending and under examination on the merits in the instant case with an effective filing date of 09/01/2022.
Claim Interpretation
The term “a live cell”, as used in claims 1-7, 13-22, and 28-30, is afforded the Broadest Reasonable Interpretation B.R.I. consistent with the specification and of one of ordinary skill in the art at the time of filing. The specification describes the “methods of nucleic acid-guided nuclease editing in cell populations, e.g., prokaryotic, archaeal, and eukaryotic cells. In some aspects, the cells include bacterial cells. In some aspects, the cells include fungal cells. In some aspects, the cells include mammalian cells” (paragraph 086). Thus, examiner will consider “a live cell” to encompass all three forms of life. The specification is silent with respect to whether “a live cell” is in vitro and/or in vivo. However, the specification only provides examples in vitro. Thus, the examiner will consider the applicant’s use of “a live cell” such as in claim 1, to be in culture only (i.e., in vitro), and not in vivo or in any animal.
The term “providing conditions” as used for example in claim 1 to allow the editing system to incorporate the desired edit or claims 14-15 to allow the cell to recover and grow is afforded the Broadest Reasonable Interpretation B.R.I. consistent with the specification and of one of ordinary skill in the art at the time of filing. Paragraphs [0121], [0123], [0124], and [0130] include conditions such as “incubation of the cells in appropriate medium and may also include providing conditions to induce transcription of an inducible promoter (e.g., adding antibiotics, adding inducers, increasing temperature)…,” but do not exclude any particular conditions. Thus, the examiner will interpret “providing conditions” consistent with the disclosure’s inclusion of certain conditions and the vast number of conditions to arrest or recover cell growth known to one of skill in the art at the time of filing, for example see Noh, M.H., Cha, S., Kim, M. et al. Recent Advances in Microbial Cell Growth Regulation Strategies for Metabolic Engineering. Biotechnol Bioproc E 25, 810–828 (2020).
Claim Objections
Claim 14 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 16. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). B.R.I. as explained above is applied to the following reasonings. Claim 14 is dependent on claim 1. Claims 1 and 14 taken together recite essentially the same steps as independent claim 16. There are 2 minor differences in wording that the examiner considers not to change the scope between claims 14 and 16. First, claim 1 recites the step “providing an editing system to a cell with a target locus…,” whereas claim 16 recites “transforming the cell” (i.e., “a cell with a target locus”) “with an editing system,” and wherein the editing systems are identical. The specification specifically recites “a variety of delivery systems may be used to introduce (e.g., transform, transfect, or transduce) nucleic acid-guided nuclease editing system components into a host cell.” Thus, “transforming” fall under claim 1’s broad language “providing an editing system.” Second, claim 14 recites “providing conditions to allow the cell to recover and grow after…,” whereas claim 16 recites “inducing the cell into a growth state.” “FIG. 1B illustrates an exemplary method for inducing and recovering a population of cells into/from a growth-arrested state for editing…,” which is applicable to both claim 14 and 16; furthermore, the specification recites that “transfer of the cells back to conditions providing an abundance of nutrients promotes a shift of the cells back to the active growth state, wherein cells are actively proliferating” ([0130]). Therefore, the scope between claims 14 and 16 is unchanged because “inducing the cell into a growth state” is accomplished by “providing conditions to allow the cell to recover and grow.”
Claim 15 objected to under 37 CFR 1.75 as being a substantial duplicate of claim 29. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). As described above, the differences between claim 14 and claim 16 regarding “providing conditions to allow the cell to recover and grow” and “inducing the cell into a growth state” are not mutually exclusive. This similarity is further illustrated by claims 15 and 29 both reciting “transferring the cell to nutrient-rich media.” Thus, the examiner is considering by “transferring the cell to nutrient-rich media,” one may accomplish either “providing conditions to allow the cell to recover and grow” or “inducing the cell into a growth state.” “
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.
Claims 1-5, 14, and 16-20, are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Doudna et. al., (US 10570418 B2, published 2/25/2020).
Regarding claims 1, 4-5, 16, and 19-20, Doudna teaches more efficient “methods of site-specific modification of a target DNA in cells of a population of eukaryotic cells” by arresting the cells in a phase where homology directed repair (HDR), i.e., a form of homologous recombination, DNA repair pathway is most active to resolve double-stand breaks (DSBs) induced during editing (see claim 1). As set forth in the instant specification, “a live cell” under B.R.I. includes eukaryotic cells (e.g., mammalian cells). Regarding claims 1 and 16 specifically, Doudna teaches “enriching” (i.e., arresting) “the population of eukaryotic cells … in a desired phase of the cell cycle, wherein the desired phase of the cell cycle comprises the M-phase” or alternatively “S-phase” of the cell cycle (claim 1 and 2), when HDR is active. Regarding claims 4-5 and 19-20 specifically, Doudna’s teachings describe the use of “chemical inhibitors” to “arrest cells at specific phases of cell cycle” before editing, for example nocodazole (Noc), which arrest cells in the G2/M phase where HDR is at its peak (see examples). Regarding claims 14 and 16 specifically, Doudna teaches that removal of the chemical stressor allows the cells to re-enter the cell cycle/growth state (column 27). Thus, Doudna teaches inducing live cells into a growth-arrested state (halted progression through the cell cycle, yet still responsive to stimuli and metabolically active) prior to editing in order to take advantage of the DNA repair pathway, HDR, that most efficiently and faithfully resolves DSBs, and then once editing has either commenced or completed, releasing the cells into a growth state.
Regarding claims 1 and 16, Doudna further teaches that the editing methods are performed using the CRISPR/Cas9 system comprising: “a Cas9 protein or a nucleic acid encoding a Cas9 protein,” “a guide RNA,” and optionally “a donor polynucleotide” (see claims 1 and 10). Doudna defines Cas9 as a CRISPR nuclease directed by a guide RNA to a target DNA sequence (i.e., a nucleic acid-guided nuclease as claimed). Doudna defines a guide RNA as “a targeting sequence that hybridizes to a target sequence of a target DNA, and a protein-binding domain that interacts with the Cas9 protein.” Doudna defines a donor polynucleotide as having homology to the target site and is used for homology-directed repair (HDR) of Cas9-induced double-strand breaks to introduce new genetic material at a specific sequence (see Columns 13-16).
Regarding claims 1-3, 14, and 16-18, Doudna further teaches that, after synchronization/arrest, the cells are then contacted with Cas9 protein, guide RNA, and donor polynucleotide under culture conditions that allow Cas9 to introduce double strand break and HDR to occur by at least 2-fold compared to unsynchronized cells; thereby, increasing efficiency of the editing and decreasing the cellular toxicity associated with DSBs before the cells are allowed to resume proliferation. Regarding claims 14 and 16 specifically, Doudna teaches the methods in live HEK293T cells, primary human neonatal fibroblast, and human embryonic stem cells that, while chemically arrested in defined cell-cycle phases, remain viable and are described as cycling like unsynchronized controls and morphologically normal as early as 1 day after nocodazole release. Regarding claims 2 and 17 specifically, such live synchronized eukaryotic cells are capable of responding to external signals, activating transcription, and producing proteins during the arrested state (see Doudna Fig1A-1D; Column 27, example 1, and rest of publication).
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.
Claims 1-7, 13-22, and 28-30 are rejected under 35 U.S.C. 103 as being unpatentable over Jinek, M. et. al., (WO 2013176772 A1, published 11/28/2013) in view of Tsakraklides V. (WO 2014182657 A1, published 11/13/2014).
With respect to claim 1 and 30, Jinek teaches: a method for performing nucleic acid-guided nuclease editing in a genome of a live cell, the method comprising: providing an editing system to a cell with a target locus, the editing system comprising: (a) (i) a nucleic acid-guided nuclease or (ii) a vector encoding the nucleic acid-guided nuclease; (b) (i) a guide RNA (gRNA) recognizing the target locus or (ii) a nucleic acid encoding the gRNA; and (c) a donor template comprising a desired edit to be incorporated into the target locus; providing conditions to allow the editing system to incorporate the desired edit into the target locus, and wherein the cell is a bacterial cell [“a method of promoting site-specific cleavage and modification of a target DNA in a cell, the method comprising introducing into the cell: (i) a DNA-targeting RNA, or a DNA polynucleotide encoding the same, wherein the DNA-targeting RNA comprises: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) a site-directed modifying polypeptide, or a polynucleotide encoding the same, wherein the site- directed modifying polypeptide comprises: (a) an RNA-binding portion that interacts with the DNA -targeting RNA; and (b) an activity portion that exhibits nuclease activity that creates a double strand break in the target DNA; wherein the site of the double strand break is determined by the DNA-targeting RNA, the contacting occurs under conditions that are permissive for nonhomologous end joining or homology-directed repair, and the target DNA is cleaved and rejoined to produce a modified DNA sequence” (see claim 92).] Jinek teaches the above method further comprises “contacting the target DNA with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA” (see claim 93). Jinek teaches “the nucleotide sequence encoding the DNA- targeting RNA is operably linked to a promoter,” and “in some cases, the promoter is an inducible promoter” (paragraph 0010). Jinek further teaches that the above method can be performed in a cell “selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, …” (see claim 95).
Jinek does not teach: arresting cells before editing, that the conditions to allow editing are provided during the growth-arrest state, the arrested cells are metabolically active and able to respond to stimuli, cell cycle blocking agents and/or growth-arrest methods, and releasing the cells from the growth-arrested state after editing.
Regarding claims 1, 16, and 30 , Tsakraklides teaches “a method of increasing the efficiency of targeted integration during genetic transformation protocols comprising the steps of synchronizing cells prior to transforming them” (see abstract and claims). Tsakraklides teaches that “transformation refers to the transfer of a nucleic acid fragment into a host organism or the genome of a host organism, resulting in genetically stable inheritance” (pg. 9 ln. 17-18), and that “any convenient technique for introducing a transgene into a microorganism can be employed in the present invention” (pg. 15 ln. 16-18).
Regarding claims 1, 2-3, 16, and 30 Tsakraklides teaches that “integration of a DNA fragment in a host genome requires action of a double-strand break (DSB) repair mechanism” and that “two major DSB repair pathways have been identified: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR)” (pg. 1 ln. 10-12). Tsakraklides teaches that while NHEJ is the predominant repair mechanism because it doesn’t require “two similar or identical molecules of DNA,” it is prone to “imprecise repair leading to loss of nucleotides” (pg. 1 ln. 10-15). However, Tsakraklides teaches that the “inventive methods increase the prevalence of homologous recombination during cell transformation, thus allowing the isolation of desired recombinant cells” without excessive screening (abstract). Tsakraklides further teaches that “HR is conserved across all three domains of life, as well as viruses, suggesting that it is a nearly universal biological mechanism” (pg. 1 ln. 27-29). Tsakraklides further teaches that whether NHEJ or HR is used to repair double-strand breaks also depends on the particular phase of the cell cycle,” and that “HR repairs DNA before a cell enters mitosis (M phase); it occurs during and shortly after DNA replication” (pg. 2 ln. 11-13). Tsakraklides teaches that “transforming cells that are in S phase with DNA carrying sequences homologous to genomic DNA increases the likelihood that the introduced DNA will integrate at the homologous locus (via HR), rather than randomly in the genome (via NHEJ). This targeting of DNA integration allows for accurate deletion or alteration of genomic information with high efficiency and without permanently altering the capacity of the organism to repair its own genome.” Tsakraklides teaches that his “method should also be applicable to increasing the efficiency of homologous recombination in extrachromosomal DNA (e.g., linear DNA, plasmids, YACs), and could be relevant in organisms with an unfavorable balance of HR to NHEJ” (pg. 5 ln. 5-13).
Regarding claim 30, Tsakraklides further teaches that his methods are applicable to “microorganisms” which include “prokaryotic and eukaryotic microbial species from the domains Bacteria and Eukaryote, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista,” and that “the terms "microbial cells" and "microbes" are used interchangeably with the term microorganism” (pg. 6 ln. 26-29). Tsakraklides expressly teaches that “E. Coli is well suited for use as the host microorganism” (pg. 10 ln. 9-10).
Regarding claims 1-7, 13-22, and 28-29, Tsakraklides further teaches a plurality of methods to arrest cells, including temperature sensitive mutants, chemically, and nutrient limitation. Tsakraklides teaches that synchronized/arrested cells can be induced into a growth state by reestablishing nutrition or removing the stressor after transformation (pgs. 16-18). Tsakraklides teaches that the promoters used in the methods “can generally be characterized as either constitutive or inducible” (pg. 12 ln. 22) and that “both types of promoters find application in the methods of the invention” (pg. 12 ln. 26). Tsakraklides further teaches that the “inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (beat or cold), or lack of nitrogen in culture media” (pg. 12 lns. 27-29). Thus, the arrested cells in Tsakraklides transformation are metabolically active and able to respond to stimuli when conditions are provided to allow the editing system to incorporate the desired edit into the target locus.
It would have been obvious to one of ordinary skill in the art to modify the CRISPR/Cas9 with donor polynucleotide/HDR template method of Jinek to include performing the editing system in growth-arrested cells, as taught by Tsakraklides, because this is a combination of known elements used for the same purpose to yield predictable results of increasing editing efficiency by leveraging a cells ability to perform HDR. Jinek already provides Cas9, guide RNA, and donor template that integrates into the target DNA via HDR or NHEJ, and expressly teaches use in a broad range of cell types, including bacterial cells. Tsakraklides shows that arresting cells before transforming them significantly increases HDR frequencies and improves fidelity and editing efficiencies during transformation. A person of ordinary skill, seeking to improve the fidelity and efficiency of the Jinek HDR method, would have been motivated to apply the same arrest/synchronization strategy of Tsakraklides to the Jinek system to favor HDR over competing repair pathways with a reasonable expectation of success.
It further would have been obvious to one of ordinary skill in the art to adapt the various growth-arrest methods demonstrated by Tsakraklides in bacterial/prokaryotic cells to the CRISPR system taught by Jinek as the transformation technique. Tsakraklides explains that HR is conserved across bacteria and eukaryotes, that the balance between NHEJ and HR depends on the cell’s growth state, and that arresting/synchronizing microbial cells (including E. Coli) by temperature shifts, chemical treatments, or nutrients limitation increases the efficiency of targeted integration via HR during transformation. In view of Jinek’s teaching that the CRISPR/Cas9 editing system with donor template can be used for site-specific modification in a wide range of cells, including bacterial cells, a person of ordinary skill in the art would have been motivated to first place bacteria cells into a growth-arrested state using Tsakraklides’s methods (for example limiting an essential nutrient), then introduce the CRISPR/Cas9 editing system with donor template while HR is favored, and finally restore the nutrients or normal conditions to allow edited cells to resume growth. Given that Tsakraklides emphasizes that HDR efficiency is enhanced when cells are in specific arrested/synchronized states and that release from arrest returns the cells to a normal state, one would have a reasonable expectation of success to implement nutrition-based arrest (or others) and subsequent nutrient re-addition (or return to normal conditions) as recited in the instant claims, in order to bias repair of Cas9-induced breaks towards HDR, effectively enhancing the fidelity and efficiency of Jinek’s CRISPR/Cas9 editing system in bacterial cells.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to COREY LANE BRETZ whose telephone number is (571)272-7299. The examiner can normally be reached M-F 9am-5pm EST.
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/COREY LANE BRETZ/Patent Examiner, 1635/1600
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635