DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s response of 02/20/2026 has been received and entered into the application file.
Claim 1 was amended in the claim set filed 02/20/2026.
Claims 26 and 27 were canceled in the claim set filed 02/20/2026.
Claims 28 and 29 were added in the claim set filed 02/20/2026.
Claims 1-7, 11-13, 16-25, and 28-29 are pending, of which claims 17-21 were previously withdrawn.
Election/Restrictions
Applicants previously elected Group I (claims 1-7, 11-13, 16, and 22-25 of the claim set filed 10/10/2025) drawn to a method of editing a target cell genome.
Claims 17-21 and 26-27 were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 26 and 27 were canceled in the amended claim set filed 02/20/2026. Election was made without traverse in the reply filed on 02/24/2025.
Accordingly, claims 1-7, 11-13, 16, 22-25, 28, and 29 are pending and under consideration.
Information Disclosure Statement
Receipt of an information disclosure statement on 02/20/2026 is acknowledged. The signed and initialed PTO-1449 has been mailed with this action.
Status of Prior Objections/Rejections
RE: Claim Rejections - 35 USC § 103
►Claims 1-4, 7, 11-12, and 16 were previously rejected under 35 U.S.C. 103 as being unpatentable over Park et al., 2020 (cited in IDS filed 11/04/2022; of record), in view of Zhang et al., 2021 (of record), Li et al., 2020 (of record), Mianné et al., 2020 (of record), and Spagnol and Dahl, 2016 (of record), as evidenced by Bertling et al., 2004 (of record), Lim et al., 2013, and Mora-Bermúdez and Ellenberg, 2006 (of record).
Applicant has traversed the rejection of record, asserting that the art does not teach the claimed method, with particular regard to the selection and expansion of individual genome-edited clones requiring at least 60% cell viability following treatment with a Class I and/or Class II histone deacetylase inhibitor. Applicant asserts that cell viability is not necessarily correlated with cell number, as cell viability is a measure of the proportion of live, healthy cells within a population and is distinct from cell number. Applicant further asserts that Applicant determined that HDAC inhibitors reduce cell viability, and thus, Applicant recognized the importance of cell viability to the presently claimed method.
In response, this is not found persuasive. As set forth in greater detail below, the impact of HDAC inhibitor treatment on cell viability has been published in the field prior to the effective filing date of the instant application. US 2016/0024474 A1 (hereinafter Conway) discloses methods of targeted genome modification of hematopoietic stem cells (abstract), said methods comprising increasing gene modification in said stem cell by culturing the cell in the presence of one or more factors that affect and/or increase stem cell expansion without loss of stemness (i.e. valproic acid (VPA)) before administration of an exogenous nuclease that mediates cleavage and/or modification of a cell’s genome (paragraph [0015]). The supplied factor may be supplied at any concentration that is sufficient to affect stem cell proliferation and/or genomic modification for the particular stem cell population (paragraph [0016]). Furthermore, Conway discloses that while VPA treatment was associated with a decrease in viability, the associated decrease in viability was very minimal before 6 days post thawing, thereby demonstrating the VPA increases stem cell multipotency marker expression with little toxicity and that VPA treatment does not affect myeloid or erythroid differentiation (paragraphs [0165] and [0166]). In other treatment cocktails, VPA treatment resulted in no loss of cell viability after extended culture, potentially due to variation from human donor to donor (paragraphs [0168]). While Conway discloses that genome modification was associated with a significant loss in viability, no decrease in viability was seen after VPA addition compared to controls until after said genome modification (paragraph [0183]). Conway further discloses that treatment with VPA enhances CRISPR/Cas induced nuclease modification when introduced before, concurrently, and/or after the CRISPR/Cas nuclease systems (paragraphs [0190] and [0191]). Therefore, Conway discloses that VPA treatment is associated with no decrease or a slight decrease in stem cell viability while increasing multipotency marker expression and maintaining myeloid or erythroid differentiation capabilities, as claimed in the instant application.
Accordingly, the amended claim limitation reciting “wherein the chromatin decondensed unmodified target cells have greater than or equal to 60% cell viability” cannot be reasonably considered to make a contribution over the prior art. Therefore, new grounds of rejection necessitated by amendment are set forth in greater detail below.
While the Examiner appreciates Applicant’s arguments regarding the claimed methodology, it nonetheless stands that the claimed workflow of treating stem cells with HDAC inhibitors to improve CRISPR editing of the same for purposes of generating therapeutically useful stem cells was, as a whole, disclosed in the art prior to the effective filing date of the instant application.
New/Maintained Grounds of Rejection
Claim Objections
Claim 29 is objected to because of the following informalities:
Claim 29 recites “the method of claim 1, further comprising karyotyping analysis of the population of clonally expanded pluripotent genome-edited cells, and wherein karyotyping analysis indicated no genomic abnormalities” (bolded emphasis added). While not strictly improper, the past tense recitation of “indicated” is not consistent with the present tense recitation of other method steps throughout the rest of the instant claim set. IT would be remedial to amend instant claim 29 such that all tenses are recited in a consistent fashion, for example by reciting “…wherein karyotyping analysis indicates no genomic abnormalities” (bolded emphasis added). This is merely an example set forth by the Examiner and is not intended to be limiting.
Appropriate correction is required.
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.
Claims 1-4, 7, 11-12, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Park et al., 2020 (hereinafter Park; cited in IDS filed 11/04/2022; of record), in view of Zhang et al., 2021 (hereinafter Zhang; of record), Li et al., 2020 (hereinafter Li; of record), Mianné et al., 2020 (hereinafter Mianné; of record), Spagnol and Dahl, 2016 (hereinafter Spagnol; of record), and US 2016/0024474 A1 (hereinafter Conway), as evidenced by Bertling et al., 2004 (hereinafter Bertling; of record), Lim et al., 2013 (hereinafter Lim; of record), and Mora-Bermúdez and Ellenberg, 2006 (hereinafter Mora-Bermúdez; of record).
With regard to amended claim 1, which recites “an ex vivo method of site-specifically editing a target cell genome, comprising treating a population of unmodified target cells with a Class I and/or Class II histone deacetylase inhibitor to provide a population of chromatin decondensed target cells, wherein the chromatin decondensed unmodified target cells have greater than or equal to 60% cell viability; live in situ nuclear imaging of the population of chromatin decondensed unmodified target cells, and quantifying the histone deacetylase inhibitor-induced decrease in chromatin condensation of the target cell nuclei, wherein the histone deacetylase inhibitor-induced decrease in chromatin condensation percentage is at least a 1% decrease in chromatin condensation percentage calculated as a percentage of heterochromatin intensity to the total nuclear intensity; introducing into the population of chromatin decondensed unmodified target cells a Cas9 ribonucleoprotein, to provide a population of site-specifically genome-edited target cells; and selecting a clone from the population of site-specifically genome-edited target cells and expanding the clone to provide a population of clonally expanded pluripotent genome-edited cells; wherein the Cas9 ribonucleoprotein comprises a Cas9 protein and a guide RNA and cleaves DNA at a cleavage site in the target cell genome, wherein the target cells comprise pluripotent cells selected from human induced pluripotent stem cells (iPSCs), human hematopoietic stem cells (HPSCs), human neural progenitor cells, human embryonic stem cells, human natural killer cells (NK cells), and human T cells, and wherein the gene expression of pluripotency genes OCT4, NANOG, SOX2, and TRA-1-60 is decreased by less than 50% in the clonally expanded genome-edited cells produced in the absence of the Class I and/or Class II histone deacetylase inhibitor” Park discloses that treatment of mouse embryonic stem cells and embryos with the histone deacetylase (HDAC) inhibitor valproic acid prior to administration of CRISPR-Cas9 gene editing machinery significantly increases CRISPR/Cas9-mediated gene editing (abstract). The methods section details this process, disclosing that mouse zygotes were collected and subsequently cultured with valproic acid until they reached the one-cell embryonic stage, at which point they were injected with pre-assembled ribonucleoproteins comprising a Cas9 protein and an sgRNA (Methods Section 2.3. Mouse Embryo Culture and Microinjection of One-Cell Stage Embryos; Figure 2; Table 1). These methods read on the instantly claimed method of treating a population of unmodified target cells with a Class I and/or Class II histone deacetylase inhibitor prior to introducing a Cas9 ribonucleoprotein comprising a Cas9 protein and a guide RNA. Per the instant disclosure, the valproic acid disclosed in Park is an exemplary Class I and/or Class II histone deacetylase inhibitor (paragraph [0016]). Additionally, the sgRNA of Park targets Cap1 for cleavage with Cas9 (Methods Section 2.1. Preparation of the RNP Complexes and Donor Vector; Results Section 3.2. Effect of VPA on CRISPR/Cas9-Mediated NHEJ in Mouse Embryos; Figure 2; Table 1), reading on the instantly claimed cleaving of the DNA at a cleavage site in the target cell genome.
However, Park does not disclose the additional limitations of amended instant claim 1, specifically the live in situ nuclear imaging, the clonal expansion step, the target cells, and the pluripotency gene expression signature. However, these deficiencies are cured by the various secondary references set forth below.
Regarding the live in situ nuclear imaging, Spagnol discloses quantification of chromatin condensation through live in situ experiments using fluorescence lifetime imaging microscopy (abstract). Specifically, Spagnol discloses measuring fluorescence intensity and quantifying mean fluorescence lifetime in cell nuclei, in which one group is treated with Trichostatin A (another HDAC inhibitor recited in instant claim 12) and another group is untreated, and subsequently comparing results from both groups (Figure 1). They found that decondensation of chromatin by treatment with Trichostatin A resulted in an increase in the mean fluorescence lifetime (page 5, paragraph 2), as well as a more uniform chromatin condensation state (page 5, paragraph 3). This live, in situ, nuclear imaging of chromatin condensation state following treatment with Trichostatin A as disclosed in Spagnol reads on the instantly claimed live, in situ, nuclear imaging of the population of chromatin decondensed unmodified target cells, and the quantification of mean fluorescence lifetime disclosed in Spagnol also reads on the instantly claimed quantification of the HDAC inhibitor-induced decrease in chromatin condensation.
Additionally, while Spagnol reports altered fluorescence intensity following Trichostatin A treatment, indicating an altered chromatin condensation state (specifically, more intense fluorescence arises from highly concentrated condensed chromatin) (Figure 1A), they quantify alterations to the chromatin condensation state using mean fluorescence lifetime rather than direct quantification of intensity. They disclose that mean fluorescence lifetime is a more accurate and sensitive method for detecting changes to chromatin state rather than intensity alone, which may restrict resolution (Page 2, Paragraph 3). That said, Mora-Bermúdez teach that despite the limitations disclosed in Spagnol, measuring changes in the local mean intensity of fluorescence in labeled chromatin regions is a simple approach to estimate chromatin compaction (Page 164-165, Section 4.4. Density analysis by fluorescence intensity measurements). Thus, while Spagnol did not quantify fluorescence intensity to detect chromatin condensation state, they did report fluorescence intensity, which could clearly be quantified and used in the methodology reported in Mora-Bermúdez.
Furthermore, treatment of cells with an HDAC inhibitor is considered to inherently result in chromatin decondensation as claimed, which is consistent with the disclosure in Spagnol. Per MPEP § 2112.02, under the principles of inherency, if the normal and usual operation of prior art “would necessarily perform the method claimed, then the method claimed will be considered to be anticipated by the prior art.” In the instant case, applicant requires HDAC treatment to result in at least a 1% decrease in chromatin condensation. Spagnol discloses significantly higher mean fluorescence lifetime (p<<0.001) in Trichostatin A-treated cells versus untreated cells (Figure 1B), indicating significant chromatin decondensation in the Trichostatin-A treated cells (Page 5, Paragraph 2). Given the disclosure of Spagnol, treatment of cells with HDAC inhibitors, such as Trichostatin A, is considered to inherently result in significant chromatin decondensation (satisfying the instant claim limitation of at least a 1% decrease in chromatin condensation), regardless of the metric by which said chromatin decondensation is reported.
While the Examiner acknowledges that the cited art does not integrate the disclosed methodology set forth above for quantification of chromatin condensation as a functional step within a genome editing protocol, the cited art does disclose quantification of chromatin condensation as a functional step within a genome editing protocol, albeit by different methodology. Park discloses quantification of acetylation and occupancy of transcription activators and chromatin remodeling complexes both prior to and following treatment with an HDAC inhibitor, stating that the observed increased acetylation and occupancy suggests that globally and significantly enhanced chromatin accessibility underlies the enhancement of CRISPR-Cas9-mediated gene editing (figure 4; section 3.4; supplementary figure 4). Thus, the disclosure of Park establishes that one of ordinary skill in the art would have been motivated to quantify acetylation and occupancy of chromatin following treatment with an HDAC inhibitor, while Spagnol and Mora-Bermúdez collectively disclose another method for quantifying chromatin state-both prior to the effective filing date of the claimed invention.
Regarding the claimed clonal expansion step, Mianné discloses a workflow to generate transgenic human pluripotent stem cell lines in which cells are edited with CRISPR reagents, isolated, and expanded as clonal populations (Figure 1). The steps of the workflow of Mianné read on the instantly claimed method. Furthermore, as disclosed in Lim, HDAC inhibitor Trichostatin A (TSA) is known to enhance iPSC differentiation as compared to iPSCs not treated with TSA, even at higher doses of 5 ng/mL (abstract; Figure 1B). As disclosed in Mianné, the therapeutic and clinical potential of pluripotent stem cells is largely due to their capacity to differentiate into any cell type (page 1, paragraph 1 of Introduction), meaning successful differentiation of pluripotent stem cells, such as the iPSCs disclosed in Lim is crucial to beginning the clonal expansion workflow reviewed in Mianné. As disclosed in Mianné, multiple strategies are known for generating, screening, and characterizing genetically engineered pluripotent stem cell lines, all of which may be manipulated by those of ordinary skill in the art to select the most appropriate strategies to reduce the time and costs to facilitate the generation of desired engineered pluripotent stem cell lines (page 2, paragraph 1; page 11, paragraph 6).
Regarding the claimed target cells, as set forth above, the disclosure of Park only encompasses mouse stem cells and embryos; whereas, the instant claim is to human stem cells. Zhang discloses a method for improving CRISPR-mediated homology-directed repair editing efficiency in human induced pluripotent stem cells (iPSCs) in which HDAC inhibitors are administered to open loci, making them accessible to CRISPR editing machinery (abstract; figures 4 and 5). Further, Zhang discloses that these cells are immortalized, allowing unlimited expansion for applications such as generating immunotherapeutic agents from patient-specific cells, which will “undoubtedly benefit” from the gene editing strategy taught therein (page 1457, column 2, paragraph 2), thereby providing a person of ordinary skill in the art explicit motivation to apply the methods taught in Zhang to clonal expansion of said edited stem cells. It is noted that the disclosure of Zhang differs from the instantly claimed method in that the HDAC inhibitors are administered after the CRISPR editing machinery is supplied (page 1459, column 2, paragraph 4-page 1460, column 1, paragraph 2). However, this disclosure nonetheless establishes that human induced pluripotent stem cells are more efficiently edited with CRISPR-Cas9 gene editing machinery when HDAC inhibitors are administered in some combination with the same in order to open loci and make them more accessible to the CRISPR-Cas9 machinery, as is instantly claimed. This is supported by the disclosure of Conway, which teaches that treatment with VPA enhances CRISPR/Cas induced nuclease modification when introduced before, concurrently, and/or after the CRISPR/Cas nuclease systems (paragraphs [0190] and [0191]).
Additionally, Zhang discloses that treatment with HDAC inhibitors did not decrease the expression of pluripotency markers OCT4, NANOG, and SOX2, suggesting treated human iPSCs maintained pluripotency and tolerated transient HDAC inhibition (page 1451, column 2, paragraph 1 under “Histone deacetylase inhibitors promote gene editing at open chromatin loci”). This is again supported by the disclosure of Conway, which teaches that VPA increases stem cell multipotency marker expression with little toxicity and that VPA treatment does not affect myeloid or erythroid differentiation (paragraphs [0165] and [0166]).
Regarding the viability of the treated cells, as set forth above, Zhang discloses methods for improving CRISPR-mediated homology-directed repair editing efficiency in human induced pluripotent stem cells (iPSCs) in which HDAC inhibitors are administered to open loci, making them accessible to CRISPR editing machinery (abstract; figures 4 and 5). While Zhang reports that human iPSC survival is not significantly impacted by VPA (at a dose of up to 2 mM) and that treatment does not attenuate pluripotency (figure S5), Zhang does not address the claimed cell viability. However, Zhang does disclose that different HDAC inhibitors have different effects on different cells, with entinostat and panobinostat having no impact on the viability of H27 and HT29 cells but considerably reducing human iPSC cell numbers (page 1458, column 2, paragraph 2). Thus, the disclosure of Zhang establishes that different HDAC inhibitors impact different cell types in different ways, thereby motivating experimentation with HDAC inhibitors to achieve the desired effect.
In support of this assertion that different HDAC inhibitors impact different cell types in different ways, the disclosure of Conway teaches that variation from human donor to donor can impact response to VPA treatment, as indicated by different VPA treatment cocktails resulting in little to no toxicity (paragraphs [0165], [0166], [0168], and [0183]). Conway further discloses that treatment with VPA both enhances CRISPR/Cas induced nuclease modification and that it may be supplied at any suitable concentration such that VPA treatment increases stem cell multipotency marker expression with little toxicity (paragraphs [0015], [0016], [0165], [0166], [0190], and [0191]). As set forth above, Conway further discloses that while genome modification was associated with a significant loss in viability, no decrease in viability was seen after VPA addition compared to controls until after said genome modification (paragraph [0183]).
Finally, regarding the claimed pluripotency gene expression signature, as set forth above, Zhang discloses a method for improving CRISPR-mediated homology-directed repair editing efficiency in human induced pluripotent stem cells (iPSCs) in which HDAC inhibitors are administered to open loci, making them accessible to CRISPR editing machinery (abstract; figures 4 and 5). While the disclosure of Zhang differs from the instantly claimed method in that the HDAC inhibitors are administered after the CRISPR editing machinery is supplied (page 1459, column 2, paragraph 4-page 1460, column 1, paragraph 2), this disclosure nonetheless establishes that human induced pluripotent stem cells are more efficiently edited with CRISPR-Cas9 gene editing machinery when HDAC inhibitors are administered in some combination with the same in order to open loci and make them more accessible to the CRISPR-Cas9 machinery, as is instantly claimed. As set forth above, given that both Zhang and Park disclose that treatment with HDAC inhibitors improves gene editing outcomes in stem cells, one of ordinary skill in the art would not reasonably predict that treatment of stem cells with HDAC inhibitors, in either order, would do anything other than improve gene editing outcomes. This is supported by the disclosure of Conway, which teaches that treatment with VPA enhances CRISPR/Cas induced nuclease modification when introduced before, concurrently, and/or after the CRISPR/Cas nuclease systems (paragraphs [0190] and [0191]).
Additionally, Zhang discloses that treatment with HDAC inhibitors did not decrease the expression of pluripotency markers OCT4, NANOG, and SOX2, suggesting treated human iPSCs maintained pluripotency and tolerated transient HDAC inhibition (page 1451, column 2, paragraph 1 under “Histone deacetylase inhibitors promote gene editing at open chromatin loci”). While Zhang is silent as to expression levels of TRA-1-60 following treatment of human iPSCs with HDAC inhibitors, given that the disclosures of Park, Zhang, Li, Mianné, and Bertling render the claimed method of amended claim 1 obvious, the functional outcomes of the claimed method and that taught in the prior art must also be obvious outcomes, as both methods perform the same steps and thus must result in the same outcome (see MPEP § 2112.02 and 2173.05(g)).
This is supported by the disclosure of Conway, which teaches that VPA increases stem cell multipotency marker expression with little toxicity and that VPA treatment does not affect myeloid or erythroid differentiation (paragraphs [0165] and [0166]).
As stated analogously in MPEP § 2112.02, if a prior art device, in its normal and usual operation, would necessarily perform the method claimed, then the method claimed will be considered to be anticipated by the prior art device. When the prior art device is the same as a device described in the specification for carrying out the claimed method, it can be assumed the device will inherently perform the claimed process. In re King, 801 F.2d 1324, 231 USPQ 136 (Fed. Cir. 1986) (The claims were directed to a method of enhancing color effects produced by ambient light through a process of absorption and reflection of the light off a coated substrate. A prior art reference to Donley disclosed a glass substrate coated with silver and metal oxide 200-800 angstroms thick. While Donley disclosed using the coated substrate to produce architectural colors, the absorption and reflection mechanisms of the claimed process were not disclosed. However, King’s specification disclosed using a coated substrate of Donley’s structure for use in his process. The Federal Circuit upheld the Board’s finding that "Donley inherently performs the function disclosed in the method claims on appeal when that device is used in ‘normal and usual operation’" and found that a prima facie case of anticipation was made out. Id. at 138, 801 F.2d at 1326. It was up to applicant to prove that Donley's structure would not perform the claimed method when placed in ambient light.)… Ex parte Novitski, 26 USPQ2d 1389 (Bd. Pat. App. & Inter. 1993) (The Board rejected a claim directed to a method for protecting a plant from plant pathogenic nematodes by inoculating the plant with a nematode inhibiting strain of P. cepacia. A U.S. patent to Dart disclosed inoculation using P. cepacia type Wisconsin 526 bacteria for protecting the plant from fungal disease. Dart was silent as to nematode inhibition but the Board concluded that nematode inhibition was an inherent property of the bacteria. The Board noted that applicant had stated in the specification that Wisconsin 526 possesses an 18% nematode inhibition rating.).
Thus, it is considered that the cited art, in combination discloses and/or motivates each and every limitation of instant claim 1.
With regard to claim 2, which recites “the cleavage site in the target cell genome is in a target cell expressed gene, a regulatory region, noncoding RNA, repeat region, integrated viral genome, topologically associating domain, or a lamina-associated domain,” as set forth above Park discloses targeting Cap1 with an sgRNA for cleavage with Cas9 in mouse embryonic stem cells and embryos (Methods Section 2.1. Preparation of the RNP Complexes and Donor Vector; Results Section 3.2. Effect of VPA on CRISPR/Cas9-Mediated NHEJ in Mouse Embryos; Figure 2; Table 1). Bertling teaches that Cap1 is strongly expressed during mouse embryonic development (Page 2326, Column 1, Paragraph 5). Thus, the gRNA cleavage target site of Park (Cap1) is a target cell expressed gene, which reads on the instantly claimed “cleavage site in the target cell genome is in a target cell expressed gene.”
With regard to claims 3 and 4, which respectively recite “the cleavage site in the target cell genome creates a precise knockout in the target cell genome” and “the precise knockout of the donor cell expressed gene proceeds primarily by a non-homologous end-joining repair pathway (NHEJ),” Park discloses that Sanger sequencing of the targeted region in mouse embryos identified insertion-deletion mutations caused by NHEJ in the Cap1 coding region (Results Section 3.2. Effect of VPA on CRISPR/Cas9-Mediated NHEJ in Mouse Embryos; Figure 2; Table 1). Given that Cap1 was targeted with the gRNA, as set forth above, detection of insertion-deletion mutations caused by NHEJ in its coding region reads on the precise knockout limitation of instant claim 3, as well as the NHEJ limitation of instant claim 4.
With regard to claim 7, which recites “the guide RNA [of instant claim 1] is a one-part sgRNA or a two-part guide RNA comprising a crRNA and a tracrRNA,” Park discloses an sgRNA targeting Cap1, which reads on the instantly claimed “one-part sgRNA.”
With regard to claim 11, which recites “a percentage of off-target genome edited sites is less than half of a percentage of on-target genome edits,” Park discloses that they did not detect any off-target mutations for Cap1 following introduction of CRISPR/Cas9 gene editing machinery, suggesting valproic acid improves on-target gene targeting with CRISPR/Cas9 in mouse embryos (Page 6, Paragraph 1). Given that no off-target mutations were detected, 100% of the genome edits were on-target and 0% of the genome edits were off-target, which reads on the instant limitations of the percentage of off-target genome edited sites being less than half of a percentage of on-target genome edits.
With regard to claim 12, which recites “the Class I and/or Class II histone deacetylase inhibitor is vorinostat, panobinostat, belinostat, entinostat, phenyl butyrate, valproic acid, trichostatin A, mocetinostat, pracinostat, dacinostat, givinostat, abexinostat, depsipeptide, or a combination thereof,” Park discloses treatment of mouse embryos and embryonic stem cells with valproic acid (an HDAC inhibitor recited in instant claim 12) prior to introducing CRISPR/Cas9 gene editing machinery (abstract), as set forth above. This disclosed treatment reads on the instantly claimed Class I and/or Class II histone deacetylase inhibitor (i.e. valproic acid). Additionally, as set forth above, Zhang discloses methods of increasing editing efficiency in human iPSCs in which CRISPR-Cas9 gene editing machinery and HDAC inhibitors are administered in combination (abstract). The HDAC inhibitors taught therein comprise vorinostat, trichostatin A, valproic acid, entinostat, and Panobinostat, as instantly claimed (page 1460, column 1, paragraph 2).
With regard to claim 16, which recites “differentiating the population of clonally expanded genome-edited cells to provide a population of differentiated genome-edited cells,” Mianné discloses that pluripotent stem cells are “valuable systems for basic research, disease-modeling, pre-clinical, and now also clinical applications” (Page 12, Paragraph 1) capable of genetic manipulation with CRISPR to build specifically-tailored transgenic lines that can then be differentiated into any cell type (Page 12, Paragraph 1). Given that instant claim 16 recites differentiating edited cells, the starting cell population must necessarily be a type of cell that isn’t fully differentiated (i.e. a stem cell or progenitor cell), meaning the pluripotent stem cells of Mianné read on the cells of instant claim 16. Additionally, the CRISPR genetic manipulation of pluripotent stem cells to establish transgenic lines for differentiation into any cell type, as disclosed in Mianné, reads on the differentiation of clonally expanded genome-edited cells of instant claim 16.
While Park discloses increased CRISPR/Cas9-mediated gene editing via the treatment of unmodified target cells (mouse embryonic stem cells and embryos) with valproic acid followed by a ribonucleoprotein comprising a Cas9 protein and an sgRNA targeting Cap1 for cleavage, they do not disclose the ex vivo nature of this method. However, Li discloses that ex vivo cell-based CRISPR/Cas9 genome editing has potential therapeutic applications (abstract) and lists several conditions currently being targeted with ex vivo CRISPR therapy (Table 1). These disclosures establish that ex vivo CRISPR/Cas9 genome editing is both possible or functional and that it has potential therapeutic applications.
This is supported by the disclosure of Conway, which teaches that the genetically modified cells produced therein may be provided as an ex vivo therapy (paragraph [0214]).
Therefore, given the utility of ex vivo CRISPR/Cas9 genome editing disclosed in Li; the increased editing success resulting from treating unmodified target cells with an HDAC inhibitor (such as valproic acid) prior to introducing Cas9 and gRNA targeting a gene of interest, as disclosed in Park; and the clonal selection and expansion workflow of edited human pluripotent stem cells disclosed in Mianné, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the methodology of Park to the clonal selection and expansion workflow of Mianné in order to perform the ex vivo CRISPR/Cas9 genome editing of Li to predictably edit the genome of unmodified target cells with greater editing success in an ex vivo manner. Per the disclosure of Zhang, someone of ordinary skill in the art would have reasonably predicted this method to function in human iPSCs, as they disclose that administration of CRISPR-Cas9 machinery and HDAC inhibitors in combination increases the editing efficiency of CRISPR-Cas9 (as is also disclosed in Conway). Furthermore, per the disclosure of Zhang, one of ordinary skill in the art would have been motivated to optimize HDAC inhibitor treatment to maintain cell viability, as disclosed in Conway. One would have been motivated to make such a modification in order to receive the expected benefit of more powerful, efficient, and effective ex vivo CRISPR/Cas9 genome editing for therapeutic purposes.
Furthermore, given the demonstrated success of methodologies to detect chromatin condensation disclosed in Spagnol, and expanded upon in Mora-Bermúdez, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to incorporate these methodologies into the gene editing system set forth above to predictably ensure that treatment with the HDAC inhibitor, as disclosed in Park and set forth above with regard to instant claim 1, was effective in decondensing chromatin to provide open chromatin exposed to the editing machinery of CRISPR/Cas9 and its associated gRNA and donor polynucleotide (as applicable). One would have been motivated to make such a modification in order to receive the expected benefit of ensuring only successfully opened chromatin is exposed to the editing machinery of CRISPR/Cas9 and its associated gRNA and donor polynucleotide (as applicable), thereby increasing both the efficacy and efficiency of the gene editing system.
Claims 5-7 are rejected under 35 U.S.C. 103 as being unpatentable over Park et al., 2020 (hereinafter Park; cited in IDS filed 11/04/2022; of record), in view of Zhang et al., 2021 (hereinafter Zhang; of record), Li et al., 2020 (hereinafter Li; of record), Mianné et al., 2020 (hereinafter Mianné ; of record), and Spagnol and Dahl, 2016 (hereinafter Spagnol; of record), and US 2016/0024474 A1 (hereinafter Conway), as evidenced by Bertling et al., 2004 (hereinafter Bertling; of record), Lim et al., 2013 (hereinafter Lim; of record), and Mora-Bermúdez and Ellenberg, 2006 (hereinafter Mora-Bermúdez; of record) as applied to claim 1 above, and further in view of Jiang and Doudna, 2017 (hereinafter Jiang; of record).
The combined disclosures of Park, Zhang, Li, Mianné, Spagnol, Conway, Bertling, Lim, and Mora-Bermúdez are described above and applied as before. However, these disclosures do not teach the donor polynucleotide comprising a synthetic DNA sequence flanked by homology arms of instant claim 5, the donor polynucleotide utility of instant claim 6, or the guide RNA composition of instant claim 7.
With regard to claim 5, which recites “the method of claim 1, further comprising introducing into the population of chromatin decondensed unmodified target cells a donor polynucleotide comprising a synthetic DNA sequence flanked by homology arms that are complementary to sequences on both sides of the cleavage target site in the target cell genome, wherein the synthetic DNA sequence in the donor polynucleotide is specifically integrated into the cleavage site of the target cell genome,” Park discloses that not only does valproic acid enhance CRISPR/Cas9-mediated gene knockout but it also enhances CRISPR/Cas9-mediated knock-in in mouse embryos (Figure 3). The targeting strategy of Park is depicted in Figure 3A, which shows the targeting construct with donor knock-in, and detailed in Methods Section 2.1. Preparation of the RNP Complexes and Donor Vector. While Park does not explicitly disclose the homology arms and synthetic DNA sequence of instant claim 5, these are inherent and necessary components for knock-in with CRISPR/Cas9, as disclosed by Jiang. Jiang teaches that CRISPR/Cas9-mediated knock-in, which proceeds via homology-directed repair, can be initiated in the presence of a donor template containing a sequence of interest (which reads on the instantly claimed synthetic DNA sequence that is integrated into the target cell genome at the specified cleavage site) flanked by homology arms (which read on the instantly claimed homology arms flanking the synthetic DNA sequence). Thus, while Park only explicitly discloses that donor vector constructs for CRISPR/Cas9-mediated knock-in were purchased from the International Mouse Phenotyping Consortium (Methods Section 2.1. Preparation of the RNP Complexes and Donor Vector), the donor vector must necessarily comprise the instantly claimed homology arms and synthetic DNA sequence, as disclosed by Jiang.
With regard to claim 6, which recites “the donor polynucleotide [of instant claim 5] includes a mutation, deletion, alteration, integration, gene correction, gene replacement, transgene insertion, nucleotide deletion, gene disruption, a gene mutation, or a combination thereof,” Park discloses that valproic acid enhances both CRISPR/Cas9-mediated gene knockout and gene knock-in in mouse embryos (Figure 3), as set forth above. While Park does not explicitly disclose the utility of their donor polynucleotide beyond insertion of a loxP site at the Cap1 locus (Figure 3A), Jiang discloses that CRISPR/Cas9-mediated gene knock-in via homology directed repair has a number of applications, including gene insertion, gene correction, and gene replacement (Page 510, Figure 2, High-fidelity HDR box). Therefore, given that Park discloses insertion of a loxP site at the Cap1 locus and that Jiang teaches that CRISPR/Cas9-mediated gene knock-in can be used for gene insertion (as in Park), gene correction, and gene replacement, the disclosure of Park reads on the instantly claimed donor polynucleotide identities, as further evidenced by the teachings of Jiang.
With regard to claim 7, which recites “the guide RNA [of instant claim 1] is a one-part sgRNA or a two-part guide RNA comprising a crRNA and a tracrRNA,” Park discloses an sgRNA targeting Cap1, which reads on the instantly claimed “one-part sgRNA.” Additionally, Jiang teaches that two-part guide RNAs are comprised of a crRNA to target the locus of interest and a tracrRNA to bind to the Cas9 endonuclease (Page 512, Paragraph 3), as instantly claimed.
Given the increased editing success (both knockout and knock-in) resulting from treating unmodified target cells with an HDAC inhibitor and quantifying the resulting chromatin decondensation prior to introducing Cas9, a gRNA targeting a gene of interest (for knockout), and a donor polynucleotide (for knock-in), as collectively disclosed in Park, Zhang, Li, Mianné, and Spagnol (as set forth above), and the requirements for and applications of CRISPR/Cas9-mediated knock-in via homology-directed repair taught in Jiang, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the homology arms flanking a sequence of interest for gene insertion, gene correction, or gene replacement into the donor polynucleotide of Park. Additionally, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to design the guide RNA of Park based on the teachings of Jiang (i.e. a one or two-part guide RNA). Both of these modifications would have predictably yielded a CRISPR/Cas9-mediated gene editing system capable of efficiently and accurately targeting a gene of interest in a cell of interest for knockout or knock-in. One would have been motivated to make such a modification in order to receive the expected benefit of facilitating efficient and accurate gene knockout using properly designed guide RNAs, as well as efficient and accurate gene knock-in using properly designed guide RNAs and donor polynucleotides.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al., 2020 (hereinafter Park; cited in IDS filed 11/04/2022; of record), in view of Zhang et al., 2021 (hereinafter Zhang; of record), Li et al., 2020 (hereinafter Li; of record), Mianné et al., 2020 (hereinafter Mianné ; of record), and Spagnol and Dahl, 2016 (hereinafter Spagnol; of record), and US 2016/0024474 A1 (hereinafter Conway), as evidenced by Bertling et al., 2004 (hereinafter Bertling; of record), Lim et al., 2013 (hereinafter Lim; of record), and Mora-Bermúdez and Ellenberg, 2006 (hereinafter Mora-Bermúdez; of record) as applied to claim 1 above, and further in view of Tóth et al., 2004 (hereinafter Tóth; as cited in the IDS filed 11/04/2022; of record) and Zentelyté et al., 2021 (hereinafter Zentelyté; of record), as evidenced by PubChem Entry: Trichostatin A.
The combined disclosures of Park, Zhang, Li, Mianné, Spagnol, Conway, Bertling, Lim, and Mora-Bermúdez are described above and applied as before. However, these disclosures do not teach the treatment conditions of unmodified target cells with trichostatin A of instant claim 13.
With regard to claim 13, which recites “the Class I and/or Class II histone deacetylase inhibitor is trichostatin A, and treating a population of unmodified target cells with the Class I and/or Class II histone deacetylase inhibitor is done for 16 to 24 hours at a temperature of 37°C, and a concentration of 3.125 ng/mL to 12.5 ng/mL,” Tóth discloses treatment with a low concentration of trichostatin A (12.5 ng/mL) at 37°C (Page 4278, Column 2, Paragraph 1) for 24 hours (Page 4278, Column 2, Paragraph 2). These treatment conditions read on the instantly claimed treatment with trichostatin A for 16 to 24 hours at 37°C and a concentration of trichostatin A ranging from 3.125 ng/mL to 12.5 ng/mL. However, Tóth does not explicitly disclose the efficacy of treating cells with this protocol.
Per MPEP § 2112.02, if the normal and usual operation of prior art “would necessarily perform the method claimed, then the method claimed will be considered to be anticipated by the prior art.” In the instant case, applicant is claiming treatment of unmodified target cells with a specific concentration of trichostatin A for a defined amount of time and at a set temperature. Given that Tóth discloses the same properties and characteristics of the instantly claimed treatment protocol, the method of Tóth is considered to read on the limitations of instant claim 13.
As Applicant has asserted, the disclosure of Tóth is drawn to the treatment of HeLa cells. In view of the disclosure of Zentelyté, however, the methods of Tóth are not considered to be limited in applicability to HeLa cells. As set forth above, Zentelyté discloses treatment of stem cells (specifically human amniotic fluid stem cells) with 20 nM of Trichostatin A (which corresponds to ~6.0474 ng/mL as calculated with the molecular weight of Trichostatin A (302.37 g/mol-see PubChem Entry: Trichostatin A)) for 24 hours to encourage differentiation of said stem cells (page 3, column 1, paragraph 4; Figure 2). As set forth above regarding instant claim 1, stem cell differentiation is crucial to their therapeutic utility (reviewed in Mianné). Therefore, given that “a person of ordinary skill in the art is also a person of ordinary creativity, not an automaton” (MPEP § 2141.03(I)), a person of ordinary skill in the art with access to the cited art would inherently be motivated to utilize the art publicly available prior to the effective filing date of the claimed invention, such as the broadly applicable cell culturing methods of Tóth and the stem cell differentiation culturing methods of Zentelyté, to advance his or her own experimental methodology and outcomes, including for therapeutic applications, as in the instant application.
Thus, while Tóth does not explicitly disclose the efficacy of treating cells with 12.5 ng/mL of trichostatin A for 24 hours at 37°C, the availability of this methodology means it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to treat targeted cells with an HDAC inhibitor to encourage gene editing with CRISPR-Cas9, as collectively disclosed by Park, Zhang, Li, Mianné, and Spagnol (as set forth above regarding instant claim 1), using the treatment conditions of Tóth to predictably produce a population of chromatin decondensed targeted cells for further editing with CRISPR/Cas9 and its associated gRNA and donor polynucleotide (as applicable). Zentelyté further supports this motivation by establishing that treatment of human stem cells with 20 nM (~6.0474 ng/mL) of Trichostatin A for 24 hours encourages stem cell differentiation, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to treat targeted stem cells with an HDAC inhibitor to encourage gene editing with CRISPR-Cas9, as collectively disclosed by Park, Zhang, Li, Mianné, and Spagnol (as set forth above regarding instant claim 1), using the Trichostatin A dosage of Zentelyté to predictably produce a population of chromatin decondensed targeted stem cells for further editing with CRISPR/Cas9 and subsequent differentiation for therapeutic applications. One would have been motivated to make such a modification in order to receive the expected benefit of producing a population of chromatin decondensed targeted cells for further editing with CRISPR/Cas9 and its associated gRNA and donor polynucleotide (as applicable), as well as of producing a differentiated population of stem cells with therapeutic potential that is suitable for the same.
Claims 22-25 are rejected under 35 U.S.C. 103 as being unpatentable over Park et al., 2020 (hereinafter Park; cited in IDS filed 11/04/2022; of record), in view of Zhang et al., 2021 (hereinafter Zhang; of record), Li et al., 2020 (hereinafter Li; of record), Mianné et al., 2020 (hereinafter Mianné ; of record), and Spagnol and Dahl, 2016 (hereinafter Spagnol; of record), and US 2016/0024474 A1 (hereinafter Conway), as evidenced by Bertling et al., 2004 (hereinafter Bertling; of record), Lim et al., 2013 (hereinafter Lim; of record), and Mora-Bermúdez and Ellenberg, 2006 (hereinafter Mora-Bermúdez; of record) as applied to claim 1 above, and further in view of Avior et al., 2016 (hereinafter Avior; of record).
The combined disclosures of Park, Zhang, Li, Mianné, Spagnol, Conway, Bertling, Lim, and Mora-Bermúdez are described above and applied as before. However, these disclosures do not teach the in vitro disease model of instant claims 22-25.
With regard to claim 22, which recites “an in vitro disease model comprising the genome-edited target cells of claim 1,” Avior discloses establishing an in vitro disease model by editing the genes of stem cells (Figure 1), which can then be cultured (Figure 1) and differentiated into the appropriate cell type for modeling the disease of interest (Page 174, Column 1, Paragraph 2), as appropriate. This disclosure reads on the instantly claimed in vitro disease model.
With regard to claim 23, which recites “the disease [modeled by the method of claim 22] is an inherited cardiac disease, Huntington's disease, Alzheimer's disease, Parkinson's disease, schizophrenia, amyotrophic lateral sclerosis, spinal muscular atrophy, Rett syndrome, Prader-Willi syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, multiple myeloma, aplastic anemia, diabetes, sickle cell disease, thalassemia, lysosomal storage diseases, Duchenne's Muscular Dystrophy, inherited retinal disorder, or cystic fibrosis, cancer, kidney disease, or a liver disease,” Avior discloses in vitro cell modeling of Parkinson’s disease (Page 174, Column 1, Paragraph 2), schizophrenia (Page 173, Column 1, Paragraph 1), amyotrophic lateral sclerosis (Page 173, Column 1, Paragraph 3), Prader-Willi syndrome (Page 174, Column 2, Paragraph 3), and cystic fibrosis (Page 175, Column 2, Paragraph 3), among others. Thus, the disclosure of Avior reads on the instantly claimed diseases to be modeled in vitro.
With regard to claim 24, which recites “the genome-edited target cells are expanded and differentiated and are alveolar epithelial cells, airway epithelial cells, neuronal cells, adipocytes, cardiomyocytes, hematopoietic cells, pancreatic beta cells, retinal epithelial cells, photoreceptors, retinal ganglion cells, epidermal cells, intestinal epithelial cells, smooth muscle cells, skeletal muscle cells, renal cells, chondrocytes, osteocytes, stromal cells, T cells, natural killer cells, macrophages, or red blood cells,” Avior discloses induced pluripotent stem cell-derived neurons for modeling Alzheimer’s (Page 173, Column 2, Paragraph 1), as well as cardiomyocytes differentiated from induced pluripotent stem cells for modeling cardiac arrhythmia disorders (Page 173, Column 2, paragraph 2). Thus, the disclosure of Avior reads on the instantly claimed expanded and differentiated target cells.
With regard to claim 25, which recites “the model [of claim 22] is used for modeling disease outcome, screening a target drug, biologic or genetic medicine treatment, or testing toxic side-effects of a treatment,” Avior discloses drug development strategies using induced pluripotent stem cells, including testing of compounds and safety assays (Figure 2), which read on the instantly claimed model for screening a target drug or testing toxic side-effects. While Avior does not explicitly disclose gene editing of these induced pluripotent stem cells in Figure 2, they disclosed gene editing of stem cells to establish an in vitro disease model in Figure 1, as set forth above.
Given the disclosed utility and efficacy of modeling diseases and their treatments in vitro using edited stem cells in Avior, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the genome-edited target cells generated by the collective methodology of Park, Zhang, Li, Mianné, and Spagnol (as set forth above with regard to instant claim 1) to predictably model diseases and their treatments in vitro, as disclosed in Avior. One would have been motivated to make such a modification in order to receive the expected benefit of producing an in vitro disease model utilizing the effectively and efficiently edited cells produced via treatment with HDAC inhibitor(s) followed by introduction of CRISPR/Cas9 machinery.
Claim 28 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al., 2020 (hereinafter Park; cited in IDS filed 11/04/2022; of record), in view of Zhang et al., 2021 (hereinafter Zhang; of record), Li et al., 2020 (hereinafter Li; of record), Mianné et al., 2020 (hereinafter Mianné ; of record), and Spagnol and Dahl, 2016 (hereinafter Spagnol; of record), and US 2016/0024474 A1 (hereinafter Conway), as evidenced by Bertling et al., 2004 (hereinafter Bertling; of record), Lim et al., 2013 (hereinafter Lim; of record), and Mora-Bermúdez and Ellenberg, 2006 (hereinafter Mora-Bermúdez; of record) as applied to claim 1 above, and further in view of Wen and Tang, 2016 (hereinafter Wen) and The International Stem Cell Initiative, 2007 (hereinafter ISCI).
The combined disclosures of Park, Zhang, Li, Mianné, Spagnol, Conway, Bertling, Lim, and Mora-Bermúdez are described above and applied as before. However, these disclosures do not teach the bulk RNA sequencing of instant claim 28.
With regard to claim 28, which recites “the method of claim 1, wherein the gene expression of pluripotency genes OCT4, NANOG, SOX2, and TRA-1-60 is determined using bulk RNA sequencing,” as set forth above, while Zhang discloses determination of pluripotency gene expression (OCT4, NANOG, and SOX2) with quantitative real-time RT-PCR (page 1460, column 2, paragraph 2), they do not disclose determination of pluripotency gene expression with bulk RNA sequencing, as instantly claimed, nor do they disclose determination of TRA-1-60 levels. However, these deficiencies are cured by Wen and ISCI.
ISCI discloses that TRA-1-60 is a pluripotency marker used to monitor human pluripotent stem cell cultures (abstract; Figure 1). Wen discloses that bulk RNA sequencing is routinely performed to analyze gene expression of thousands or even millions of cells (abstract; page 1, column 1, paragraph 1). While Wen discloses that single-cell RNA sequencing is capable of producing a comprehensive view of the heterogeneity of a complex population of cells and even identifying numerous sub-populations of cells missed in bulk RNA-sequencing (page 2, column 1, paragraph 3; page 3, column 1, paragraph 1), Wen also discloses that bulk RNA sequencing is the standard methodology for analyzing gene expression in a sample of cells with greater coverage and lower noise/amplification errors than that achieved with single-cell sequencing (abstract; page 1, column 1, paragraph 1; page 7, column 2, paragraph 1; page 8, column 2, paragraph 4).
Given that Park, Zhang, Li, Mianné, Spagnol, Conway, Bertling, Lim, and Mora- Bermúdez collectively disclose the method of claim 1 (as set forth above), wherein Zhang specifically discloses determination of pluripotency gene expression as part of the workflow taught therein, that Wen discloses that bulk RNA sequencing is the standard methodology for analyzing gene expression in a sample of cells with greater coverage and lower noise/amplification errors than that achieved with single-cell sequencing, and that ISCI discloses that TRA-1-60 is a pluripotency marker used to monitor human pluripotent stem cell cultures, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to monitor the pluripotency of treated stem cells (as disclosed in Zhang) via bulk RNA sequencing (as disclosed in Wen) to predictably monitor pluripotency gene expression (including TRA-1-60, as disclosed in ISCI) and thus stem cell pluripotency status (as disclosed in ISCI). One would have been motivated to make such a modification in order to receive the expected benefit of monitoring pluripotency gene expression in treated stem cells.
Claim 29 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al., 2020 (hereinafter Park; cited in IDS filed 11/04/2022; of record), in view of Zhang et al., 2021 (hereinafter Zhang; of record), Li et al., 2020 (hereinafter Li; of record), Mianné et al., 2020 (hereinafter Mianné ; of record), and Spagnol and Dahl, 2016 (hereinafter Spagnol; of record), and US 2016/0024474 A1 (hereinafter Conway), as evidenced by Bertling et al., 2004 (hereinafter Bertling; of record), Lim et al., 2013 (hereinafter Lim; of record), and Mora-Bermúdez and Ellenberg, 2006 (hereinafter Mora-Bermúdez; of record) as applied to claim 1 above, and further in view of Rayner et al., 2019 (hereinafter Rayner) and Papathanasiou et al., 2021 (hereinafter Papathanasiou).
The combined disclosures of Park, Zhang, Li, Mianné, Spagnol, Conway, Bertling, Lim, and Mora-Bermúdez are described above and applied as before. However, these disclosures do not teach the karyotyping analysis of instant claim 29.
With regard to claim 29, which recites “the method of claim 1, further comprising karyotyping analysis of the population of clonally expanded pluripotent genome-edited cells, and wherein karyotyping analysis indicated no genomic abnormalities,” Rayner discloses that CRISPR-Cas9 editing is prone to causing off-target mutations, chromosomal abnormalities, and large mutations (abstract; page 406, column 1, paragraph 2-page 407, column 1, paragraph 1). Therefore, CRISPR-Cas9 clones should be tested for large-scale deletions and disruptions via cytogenetic analyses including karyotyping (page 407, column 1, paragraph 3; page 408, column 1, paragraph 3; Figure 1). Rayner discloses that analysis of CRISPR clones indicated that while the expected mutations were introduced by CRISPR/Cas9 editing (Figure 2), the CRISPR clones also exhibited chromosomal instability (Figure 3). While the disclosure of Rayner is primarily drawn to cancer cell analysis, Rayner nonetheless teaches a cost-effective visual method for assessing chromosomal rearrangements and large deletions in CRISPR-Cas9 mutated clones (page 413, column 2, paragraph 2; page 415, column 2, paragraph 4). The findings of Rayner are supported by Papathanasiou, which discloses that CRISPR-Cas9 editing induced karyotype aberrations during embryonic development, thereby demonstrating that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosomal structural alterations (abstract). Thus, the findings of Rayner are not limited to cancer cells, and CRISPR-Cas9 editing is known to induce karyotype aberrations in multiple cell types, which may be detected in a cost-effective manner via karyotyping.
Given that Park, Zhang, Li, Mianné, Spagnol, Conway, Bertling, Lim, and Mora- Bermúdez collectively disclose the method of claim 1 (as set forth above), and that Rayner and Papathanasiou disclose that CRISPR-Cas9 editing is known to induce karyotype aberrations in multiple cell types, which may be detected in a cost-effective manner via karyotyping, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to analyze the CRISPR-Cas9 edited cells produced via the methods of claim 1 via karyotyping to predictably detect unwanted on-target side effects, including major chromosomal structural alterations (as disclosed in Rayner and Papathanasiou). One would have been motivated to make such a modification in order to receive the expected benefit of detecting unwanted on-target side effects, including major chromosomal structural alterations.
Conclusion
No claims are allowed.
Claim 29 is objected to.
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 Sarah E Allen whose telephone number is (571)272-0408. The examiner can normally be reached M-Th 8-5, F 8-12.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637
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