GENERATION OF INDUCED PLURIPOTENT CELLS BY CRISPR ACTIVATION

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 . Claims 15-41, of record 10/14/2024, are pending and subject to prosecution. Priority The instant application is a DIV of 16/957086 (US patent 12146166, filed 6/22/2020), which is a national stage entry of PCT/US2018/067679 (filed 12/27/2018), which claims benefit of provisional application 62/611202 (filed 12/28/2017). Drawings Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification: The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2). Specification The use of the terms Parnate, FuGENE, Polybrene, B-27, Neurobasal, MACSQuant, FlowJo, RNeasy, QIAshredder, DNA-free, SYBR, Prism, EZ-ChIP, glutaGRO, Lipofectamine, KnockOut, Accutase, iScript, EVOS, TaqMan, StepOnePlus, QuantStudio, StepOne, and GraphPad, which are trade names or marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore, the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms. Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks. Claim Objections Claim 27 is objected to because of the following informalities: In line 1 of claim 27, “two” should be inserted after “the at least”. Appropriate correction is required. 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. The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows: 1. Determining the scope and contents of the prior art. 2. Ascertaining the differences between the prior art and the claims at issue. 3. Resolving the level of ordinary skill in the pertinent art. 4. Considering objective evidence present in the application indicating obviousness or nonobviousness. 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. Claims 15-16, 18-19, 22, and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Balboa et al. (Stem Cell Reports, 2015), of record in IDS dated 9/8/2026, in view of Hilton et al. (Nature Biotechnology, 2015), of record in IDS dated 9/8/2026. Regarding claims 15-16, 18, and 22: Balboa et al. teach the generation of iPSCs using a CRISPR activation system comprising dCas9 fused to VP16 multimers and sgRNA (See fig. 1A and 1D). Multiple gRNAs specific for the endogenous Oct4 promoter (See page 449, col. 1, full ¶2 and page 451, col. 1, full ¶2) were introduced into skin fibroblasts (which read on “a non-iPSC”) via plasmid (which reads on “a polynucleotide”), also encoding dCas9 and gRNAs targeting the additional pluripotency genes Sox2, Nanog, and Lin28 (See page 451, col. 2, ¶1). Balboa et al. teach activation of transcription with dCas9 fused to VP16 repeats and do not expressly teach a dCas9 protein fused to a SunTag. However, Balboa et al. suggest that the size of the dCas9 activator construct could be reduced by incorporating a SunTag peptide tail (See page 455, col. 1, full ¶1), which would motivate one of ordinary skill to make this modification. Balboa et al. do not teach attachment of a p300 core to the SunTag array or targeting the Oct4 promoter and an Oct4 enhancer with gRNAs. Hilton et al. teach CRISPR activation of Oct4 expression by dCas9 fused to the p300 core and multiple gRNAs targeting the Oct4 promoter, proximal enhancer, or distal enhancer (See Abstract and fig. 2). It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Balboa et al. to comprise a p300 core linked to dCas9 and a SunTag array. One would have been motivated to make this modification because Hilton et al. teach that the p300 core catalyzes H3K27 acetylation at target sites that leads to robust transcriptional activation from promoters and enhancers (See Abstract). There would be a reasonable expectation of success in doing so because Hilton et al. demonstrate that the p300 core retains acetyltransferase activity as a fusion protein (See fig. 1 and 6). It also would have been obvious to use gRNAs targeting the Oct4 promoter and one or both enhancers in the composition. Combining prior art elements according to known methods to yield predictable results is considered to be prima facie obvious. See MPEP 2143(I)(A). Hilton et al. demonstrate that dCas9-p300 core and gRNAs are able to activate transcription via H3K27 acetylation when targeted to the Oct4 promoter and enhancers individually (See fig. 2), and one of ordinary skill in the art could reasonably expect histone acetylation to occur at the promoter and enhancer(s) when targeted together and reasonably expect such epigenetic modification to result in Oct4 transcription. Regarding claim 19: Following the discussion of claims 15-16, 18, and 22, Balboa et al. do not teach a gRNA comprising a sequence corresponding to SEQ ID NOs 1-6 or 57-58. Hilton et al. teach targeting of the Oct4 promoter with a gRNA comprising a sequence identical to instant SEQ ID NO 58 (See supplementary table 4 and alignment below). PNG media_image1.png 138 726 media_image1.png Greyscale It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to further modify the method of Balboa et al., modified by Hilton et al., to substitute the Oct4 promoter-targeting gRNA taught by Hilton et al. Substitution of one known element for another known element is considered to be prima facie obvious, absent a showing that the substitution yields more than predictable results. See MPEP 2143(I)(B). Regarding claim 24: Following the discussion of claims 15-16, 18, and 22, Balboa et al. do not expressly teach the inclusion of doxycycline for generating iPSCs. However, Balboa et al. teach expression of dCas9 from a plasmid having a doxycycline-inducible promoter (See page 451, col. 1, full ¶2). It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to further modify the method of Balboa et al., modified by Hilton et al. and Tanenbaum et al., to use a doxycycline-inducible promoter to express dCas9 and/or gRNAs, thereby producing a composition comprising doxycycline. One would be motivated to make this modification because Balboa et al. indicate that such a promoter enables conditional expression without leakiness (See page 451, col. 1, full ¶2), and this modification could be readily made. Regarding claim 25: Following the discussion of claims 15-16, 18, and 22, Balboa et al. teach that CRISPR-modified cells expressed Sox2 while control fibroblasts did not (which reads on “increased expression of… Sox2… as compared to the non-iPSC”) (See fig. 2C). Claims 15-16, 18-19, 21-22, and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Balboa et al. (Stem Cell Reports, 2015), of record, in view of Hilton et al. (Nature Biotechnology, 2015), of record, further in view of Zhang et al. (US 20140186958 A1). The teachings of Balboa et al. and Hilton et al. are set forth in the rejection above and are incorporated herein in their entirety. Regarding claim 21: Following the discussion of claims 15-16, 18-19, 22, and 24-25, Balboa et al., modified by Hilton et al., render obvious the generation of iPSCs using a CRISPR system wherein the Sox2 gene is activated but do not teach targeting of a Sox2 with a gRNA comprising the sequence of SEQ ID NOs 12-21 or 59. Zhang et al. teach a gRNA for targeting the human Sox2 locus comprising a sequence identical to instant SEQ ID NO 59 (See ¶0054, fig. 12, SEQ ID NO 45, and alignment below). PNG media_image2.png 136 726 media_image2.png Greyscale It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to further modify the method of Balboa et al., modified by Hilton et al., to substitute the Sox2-targeting gRNA taught by Zhang et al. Substitution of one known element for another known element is considered to be prima facie obvious, absent a showing that the substitution yields more than predictable results. See MPEP 2143(I)(B). Claims 15-16, 18-19, and 22-25 are rejected under 35 U.S.C. 103 as being unpatentable over Balboa et al. (Stem Cell Reports, 2015), of record, in view of Hilton et al. (Nature Biotechnology, 2015), of record, further in view of Valamehr et al. (US 20170073643 A1). The teachings of Balboa et al. and Hilton et al. are set forth in the rejection above and are incorporated herein in their entirety. Regarding claim 23: Following the discussion of claims 15-16, 18-19, 22, and 24-25, Balboa et al., modified by Hilton et al., render obvious the generation of iPSCs using a CRISPR activation system but do not teach the use of TGFβR, GSK3, MEK, and ROCK inhibitors. Valamehr et al. teach methods of producing pluripotent cells, such as iPSCs (See Abstract and ¶0022). In embodiments, reprogramming can comprise contacting the non-pluripotent cells with a GSK3 inhibitor, a MEK inhibitor, a TGFβR inhibitor, and a ROCK inhibitor, which can be small molecules (See ¶0065, 0098, 0127, 0209, and 0357). The inhibitors can be used in conjunction with the introduction of reprogramming factors into cells (See ¶0357). It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to further modify the method of Balboa et al., modified by Hilton et al., to comprise the addition of a GSK3 inhibitor, a MEK inhibitor, a TGFβR inhibitor, and a ROCK inhibitor during cell reprogramming to iPSCs. One would have been motivated to make this modification because Valamehr et al. indicate that media comprising these components enable efficient reprogramming (See ¶0272). There would be a reasonable expectation of success in doing so because the inhibitors could be readily added to the cell culture medium. Claims 15-19, 22, 24-29, 32-38, and 40-41 are rejected under 35 U.S.C. 103 as being unpatentable over Balboa et al. (Stem Cell Reports, 2015), of record, in view of Hilton et al. (Nature Biotechnology, 2015), of record, further in view of Li et al. (Proceedings of the National Academy of Sciences, 2012). The teachings of Balboa et al. and Hilton et al. are set forth in the rejection above and are incorporated herein in their entirety. Regarding claims 17, 26-29, 32-34, 36-38, and 40-41: Following the discussion of claims 15-16, 18-19, 22, and 24-25, Balboa et al., modified by Hilton et al., render obvious the generation of iPSCs using a CRISPR activation system but do not teach an increased number of iPSCs from targeting the Oct4 promoter and an Oct4 enhancer compared to targeting only the promoter. Li et al. teach Oct4-activating small molecule compounds that increased reprogramming efficiency and enhanced iPSC formation when introduced with reprogramming factors including Oct4 (See Abstract). The compounds increased expression of Oct4 in mouse embryonic fibroblasts and increased the number of colonies with stem cell-like morphology (See fig. 2). It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention that targeting both the Oct4 promoter and an enhancer with gRNAs for CRISPR activation could be used to increase the number of iPSCs generated. Hilton et al. teach that a pool of gRNAs targeting either the Oct4 promoter or the proximal or distal enhancer significantly increase Oct4 transcription when used with dCas9-p300 core (See fig. 2B). One of ordinary skill could reasonably expect enrichment of H3K27 acetylation (which reads on “remodeling an endogenous locus”) at each of the targeted sites to further promote gene activation when combined, and because Li et al. correlate Oct4 expression levels with iPSC colony number (See fig. 2), an increase in the iPSC population could be reasonably expected through targeting the Oct4 promoter and enhancer(s) for activation. Regarding claim 35: Following the discussion of claims 15-19, 22, 24-29, 32-34, 36-38, and 40-41, Balboa et al. teach dCas9 constructs with VP16 repeats for activating Oct4 expression (See fig. 1A), and Hilton et al. teach that dCas9 fused to the VP16 tetramer VP64 can be used activate Oct4 transcription (See fig. 2B). One of ordinary skill in the art would have therefore found it obvious to substitute VP64 as the dCas9-associated transcription activator. Substitution of one known element for another known element is considered to be prima facie obvious, absent a showing that the substitution yields more than predictable results. See MPEP 2143(I)(B). Claims 15-19, 22, 24-29, 31-38, and 40-41 are rejected under 35 U.S.C. 103 as being unpatentable over Balboa et al. (Stem Cell Reports, 2015), of record, in view of Hilton et al. (Nature Biotechnology, 2015), of record, further in view of Li et al. (Proceedings of the National Academy of Sciences, 2012), further in view of Zhang et al. (US 20140186958 A1). The teachings of Balboa et al., Hilton et al., Li et al., and Zhang et al. are set forth in the rejection above and are incorporated herein in their entirety. Regarding claim 31: Following the discussion of claims 15-19, 22, 24-29, 32-38, and 40-41, Balboa et al., modified by Hilton et al. and Li et al., render obvious the generation of iPSCs using a CRISPR system wherein the Sox2 gene is activated but do not teach targeting of a Sox2 with a gRNA comprising the sequence of SEQ ID NOs 12-21 or 59. Zhang et al. teach a gRNA for targeting the human Sox2 locus comprising a sequence identical to instant SEQ ID NO 59 (See ¶0054, fig. 12, SEQ ID NO 45, and alignment below). PNG media_image2.png 136 726 media_image2.png Greyscale It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to further modify the method of Balboa et al., modified by Hilton et al. and Li et al., to substitute the Sox2-targeting gRNA taught by Zhang et al. Substitution of one known element for another known element is considered to be prima facie obvious, absent a showing that the substitution yields more than predictable results. See MPEP 2143(I)(B). Claims 15-19, 22, 24-29, and 32-41 are rejected under 35 U.S.C. 103 as being unpatentable over Balboa et al. (Stem Cell Reports, 2015), of record, in view of Hilton et al. (Nature Biotechnology, 2015), of record, further in view of Li et al. (Proceedings of the National Academy of Sciences, 2012), further in view of Valamehr et al. (US 20170073643 A1). The teachings of Balboa et al., Hilton et al., Li et al., and Valamehr et al. are set forth in the rejection above and are incorporated herein in their entirety. Regarding claim 39: Following the discussion of claims 15-19, 22, 24-29, 32-38, and 40-41, Balboa et al., modified by Hilton et al. and Li et al., render obvious the generation of iPSCs using a CRISPR activation system but do not teach the use of TGFβR, GSK3, MEK, and ROCK inhibitors. Valamehr et al. teach methods of producing pluripotent cells, such as iPSCs (See Abstract and ¶0022). In embodiments, reprogramming can comprise contacting the non-pluripotent cells with a GSK3 inhibitor, a MEK inhibitor, a TGFβR inhibitor, and a ROCK inhibitor (See ¶0065, 0098, and 0127). The inhibitors can be used in conjunction with the introduction of reprogramming factors into cells (See ¶0357). It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to further modify the method of Balboa et al., modified by Hilton et al. and Li et al., to comprise the addition of a GSK3 inhibitor, a MEK inhibitor, a TGFβR inhibitor, and a ROCK inhibitor during cell reprogramming to iPSCs. One would have been motivated to make this modification because Valamehr et al. indicate that media comprising these components enable efficient reprogramming (See ¶0272). There would be a reasonable expectation of success in doing so because the inhibitors could be readily added to the cell culture medium. Allowable Subject Matter Claims 20 and 30 are objected to as being dependent upon a rejected base claim but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims. The following is a statement of reasons for the indication of allowable subject matter: A composition comprising Oct4 enhancer-targeting sgRNAs with sequences corresponding to instant SEQ ID NOs 7-11 appear to be free of the prior art. Conclusion Any inquiry concerning this communication or earlier communications from the examiner should be directed to JENNIFER S SPENCE, whose telephone number is 571-272-8590. The examiner can normally be reached M-F 8:30-5:30. 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, Christopher M Babic, can be reached at 571-272-8507. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. 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